Method of preventing adverse effects by GLP-1

ABSTRACT

A method for preventing or reducing adverse effects such as profuse sweating, nausea and vomiting, which normally are associated with subcutaneous and intravenous administration of glucagon-like peptide 1 (GLP-1) therapy is provided. In particular, the method comprises the rapid administration of a GLP-1 formulation into the pulmonary circulation such as by inhalation, directly into pulmonary alveolar capillaries using a dry powder drug delivery system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Nos. 60/982,368 filed Oct. 24, 2007; 60/985,620filed Nov. 5, 2007; 61/033,740 filed Mar. 4, 2008; and 61/052,127 filedMay 9, 2008. The entire contents of each of these applications areincorporated by reference herein.

TECHNICAL FIELD

Disclosed herein is a method for preventing or reducing adverse effectssuch as profuse sweating, nausea and vomiting, which normally areassociated with the subcutaneous and intravenous administration ofglucagon-like peptide 1 (GLP-1) therapy. In particular, the methodcomprises the administration of GLP-1 into the pulmonary circulationsuch as by inhalation into pulmonary alveolar capillaries using a drypowder drug delivery system.

BACKGROUND

Drug delivery systems for the treatment of disease which introduceactive ingredients into the circulation are numerous and include oral,transdermal, subcutaneous and intravenous administration. While thesesystems have been used for quite a long time and can deliver sufficientmedication for the treatment of many diseases, there are numerouschallenges associated with these drug delivery mechanisms. Inparticular, the delivery of effective amounts of proteins and peptidesto treat a target disease has been problematic. Many factors areinvolved in introducing the right amount of the active agent, forexample, preparation of the proper drug delivery formulation so that theformulation contains an amount of active agent that can reach its targetsite(s) of action in an effective amount.

The active agent must be stable in the drug delivery formulation and theformulation should allow for absorption of the active agent into thecirculation and remain active so that it can reach the site(s) of actionat effective therapeutic levels. Thus, in the pharmacological arts, drugdelivery systems which can deliver a stable active agent are of utmostimportance.

Making drug delivery formulations therapeutically suitable for treatingdisease, depends on the characteristics of the active ingredient oragent to be delivered to the patient. Such characteristics can includein a non-limiting manner solubility, pH, stability, toxicity, releaserate, and ease of removal from the body by normal physiologic processes.For example, in oral administration, if the agent is sensitive to acid,entericcoatings have been developed using pharmaceutically acceptablematerials which can prevent the active agent from being released in thelow pH (acid) of the stomach. Thus, polymers that are not soluble atacidic pH are used to formulate and deliver a dose containingacid-sensitive agents to the small intestine where the pH is neutral. Atneutral pH, the polymeric coating can dissolve to release the activeagent which is then absorbed into the enteric systemic circulation.Orally administered active agents enter the systemic circulation andpass through the liver. In certain cases, some portion of the dose ismetabolized and/or deactivated in the liver before reaching the targettissues. In some instances, the metabolites can be toxic to the patient,or can yield unwanted side effects.

Similarly, subcutaneous and intravenous administration ofpharmaceutically-active agents is not devoid of degradation andinactivation. With intravenous administration of drugs, the drugs oractive ingredients can also be metabolized, for example in the liver,before reaching the target tissue. With subcutaneous administration ofcertain active agents, including various proteins and peptides, there isadditionally degradation and deactivation by peripheral and vasculartissue enzymes at the site of drug delivery and during travel throughthe venous blood stream. In order to deliver a dose that will yield anacceptable quantity for treating disease with subcutaneous andintravenous administration of an active agent, dosing regimes willalways have to account for the inactivation of the active agent byperipheral and vascular venous tissue and ultimately the liver.

SUMMARY

Disclosed herein is a method for preventing or reducing adverse effectssuch as profuse sweating, nausea and vomiting, which normally areassociated with the subcutaneous and intravenous administration ofglucagon-like peptide 1 (GLP-1) therapy. In particular, the methodcomprises the administration of GLP-1 into the pulmonary circulationsuch as by inhalation into pulmonary alveolar capillaries using a drypowder drug delivery system.

In one embodiment, a method is provided for the treatment ofhyperglycemia and/or diabetes in a patient, comprising the step ofadministering prandially to a patient in need of treatment an inhalabledry powder formulation, comprising a therapeutically effective amount ofa GLP-1 molecule; wherein the administration does not result in at leastone side effect selected from the group consisting of nausea, vomitingand profuse sweating.

In another embodiment, the patient is a mammal suffering with Type 2diabetes mellitus. In another embodiment, the GLP-1 formulationcomprises about 0.5 mg to about 3 mg of GLP-1 in the formulation. In yetanother embodiment, the inhalable dry powder formulation furthercomprises a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

In one embodiment, a method is provided for reducing glucose levels in aType 2 diabetic patient suffering with hyperglycemia, the methodcomprising the step of administering to the patient in need of treatmentan inhalable dry powder formulation for pulmonary administrationcomprising a therapeutically effective amount of GLP-1, and adiketopiperazine or pharmaceutically acceptable salt thereof.

In another embodiment, the inhalable dry powder formulation comprises adiketopiperazine. In another embodiment, the diketopiperazine is2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl; or apharmaceutically acceptable salt thereof.

In another embodiment, the GLP-1 molecule is selected from the groupconsisting of a native GLP-1, a GLP-1 metabolite, a GLP-1 derivative, along acting GLP-1, a GLP-1 mimetic, an exendin, or an analog thereof, orcombinations thereof.

In another embodiment, the method further comprises administering to apatient a therapeutically amount of an insulin molecule. In anotherembodiment, the inhalable dry powder formulation comprises the GLP-1molecule co-formulated with the insulin molecule. In yet anotherembodiment, the insulin molecule is administered separately as aninhalable dry powder formulation. In another embodiment the insulin is arapid acting or a long acting insulin.

In another embodiment, the method further comprises administering aformulation comprising a long acting GLP-1 analog.

In another embodiment, the inhalable dry powder formulation lacksinhibition of gastric emptying.

In another embodiment, the glucose levels are reduced by from about 0.1mmol/L to about 3 mmol/L for a period of approximately four hours afteradministration of the inhalable formulation to the patient. In anotherembodiment, the inhalable formulation is administered to the Type 2diabetic patient prandially, preprandially, prandially, post-prandiallyor in a fasting state. In another embodiment, the GLP-1 formulationcomprises from about 0.02 mg to about 2 mg of GLP-1 in the formulation.

In one embodiment, a kit is provided for the treatment of diabetesand/or hyperglycemia comprising: a) a medicament cartridge operablyconfigured to fit into a dry powder inhaler and containing a dry powderformulation comprises a GLP-1 molecule, and a diketopiperazine of theformula: 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X isselected from the group consisting of succinyl, glutaryl, maleyl, andfumaryl, or salt thereof, and b) an inhalation device operablyconfigured to receive/hold and securely engage the cartridge.

In another embodiment, a kit is provided for the treatment ofhyperglycemia in a type 2 diabetic patient, which comprises a pulmonarydrug delivery system, comprising: a) a medicament cartridge operablyconfigured to fit into a dry powder inhaler and containing a dry powderformulation comprises a GLP-1 molecule, and a diketopiperazine of theformula: 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X isselected from the group consisting of succinyl, glutaryl, maleyl, andfumaryl, or salt thereof, and b) an inhalation device operablyconfigured to adapt and securely engage the cartridge.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean plasma concentration of active glucagon-likepeptide 1 (GLP-1) in subjects treated with an inhalable dry powderformulation containing a GLP-1 dose of 1.5 mg measured at various timesafter inhalation.

FIG. 2A depicts the mean plasma concentration of insulin in subjectstreated with an inhalable dry powder formulation containing a GLP-1 doseof 1.5 mg measured at various times after inhalation.

FIG. 2B depicts the plasma concentration of GLP-1 in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation compared to subjectstreated with a subcutaneous administration of GLP-1.

FIG. 2C depicts the plasma insulin concentration in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation compared to subjectstreated with an intravenous GLP-1 dose of 50 μg and subjects treatedwith a subcutaneous GLP-1 dose.

FIG. 3 depicts the mean plasma concentration of the C-peptide insubjects treated with an inhalable dry powder formulation containing aGLP-1 dose of 1.5 mg measured at various times after inhalation.

FIG. 4 depicts the mean plasma concentration of glucose in subjectstreated with an inhalable dry powder formulation containing GLP-1 dosesof 0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg, measured at varioustimes after inhalation.

FIG. 5 depicts mean plasma insulin concentrations in patients treatedwith an inhalable dry powder formulation containing GLP-1 doses of 0.05mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg. The data shows that insulinsecretion in response to pulmonary GLP-1 administration is dosedependent.

FIG. 6 depicts mean plasma glucagon concentrations in patients treatedwith an inhalable dry powder formulation containing GLP-1 doses of 0.05mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg.

FIG. 7 depicts the mean plasma exendin concentrations in male ZuckerDiabetic Fat (ZDF) rats receiving exendin-4/FDKP (fumaryldiketopiperazine) powder by pulmonary insufflation versus subcutaneous(SC) administered exendin-4. The closed squares represent the responsefollowing pulmonary insufflation of exendin-4/FDKP powder. The opensquares represent the response following administration of SC exendin-4.Data are plotted as means±SD.

FIG. 8 depicts changes in blood glucose concentration from baseline inmale ZDF rats receiving either air control, exendin-4/FDKP powder, orGLP-1/FDKP powder by pulmonary insufflation versus subcutaneouslyadministered exendin-4. The graph also shows a combination experiment inwhich the rats were administered by pulmonary insufflation an inhalationpowder comprising GLP-1/FDKP, followed by an inhalation powdercomprising exendin-4/FDKP. In the graph, the closed diamonds representthe response following pulmonary insufflation of exendin-4/FDKP powder.The closed circles represent the response following administration ofsubcutaneous exendin-4. The triangles represent the response followingadministration of GLP-1/FDKP powder. The squares represent the responsefollowing pulmonary insufflation of air alone. The stars represent theresponse given by 2 mg of GLP-1/FDKP given to the rats by insufflationfollowed by a 2 mg exendin-4/FDKP powder administered also byinsufflation.

FIG. 9A depicts the mean plasma oxyntomodulin concentrations in male ZDFrats receiving oxyntomodulin/FDKP powder by pulmonary insufflationversus intravenous (IV) oxyntomodulin. The squares represent theresponse following IV administration of oxyntomodulin alone. The uptriangles represent the response following pulmonary insufflation of 5%oxyntomodulin/FDKP powder (0.15 mg oxyntomodulin). The circles representthe response following pulmonary insufflation of 15% oxyntomodulin/FDKPpowder (0.45 mg oxyntomodulin). The down triangles represent theresponse following pulmonary insufflation of 30% oxyntomodulin/FDKPpowder (0.9 mg oxyntomodulin). Data are plotted as means±SD.

FIG. 9B depicts the cumulative food consumption in male ZDF ratsreceiving 30% oxyntomodulin/FDKP powder (0.9 mg oxyntomodulin) bypulmonary insufflation (1); oxyntomodulin alone (1 mg oxyntomodulin) byIV injection (2); or air control (3).

FIG. 10A depicts the mean plasma oxyntomodulin concentrations in maleZDF rats receiving oxyntomodulin/FDKP powder by pulmonary insufflationversus air control. The squares represent the response followingadministration of air control. The circles represent the responsefollowing pulmonary insufflation of oxyntomodulin/FDKP powder (0.15 mgoxyntomodulin). The up triangles represent the response followingpulmonary insufflation of oxyntomodulin/FDKP powder (0.45 mgoxyntomodulin). The down triangles represent the response followingpulmonary insufflation of oxyntomodulin/FDKP powder (0.9 mgoxyntomodulin). Data are plotted as means±SD.

FIG. 10B depicts data from experiments showing cumulative foodconsumption in male ZDF rats receiving 30% oxyntomodulin/FDKP powder atvarying doses including 0.15 mg oxyntomodulin (1); 0.45 mg oxyntomodulin(2); or 0.9 mg oxyntomodulin (3) by pulmonary insufflation compared toair control (4). Data are plotted as means±SD. An asterisk (*) denotesstatistical significance.

FIG. 11 depicts the glucose values obtained from six fasted Type 2diabetic patients following administration of a single dose of aninhalable dry powder formulation containing GLP-1 at various timepoints.

FIG. 12 depicts the mean glucose values for the group of six fasted Type2 diabetic patients of FIG. 11, in which the glucose values areexpressed as the change of glucose levels from zero time (dosing) forall six patients.

FIG. 13 depicts data obtained from experiments in which ZDF rats wereadministered exendin-4 in a formulation comprising a diketopiperazine ora salt of a diketopiperazine, wherein the exendin-4 was provided byvarious routes of administration (liquid installation (LIS), SC,pulmonary insufflation (INS)) in an intraperitoneal glucose tolerancetest (IPGTT). In one group, rats were treated with exendin-4 incombination with GLP-1 by pulmonary insufflation.

FIG. 14 depicts cumulative food consumption in male ZDF rats receivingair control by pulmonary insufflation, protein YY(3-36) (PYY) alone byIV injection, PYY alone by pulmonary instillation, 10% PYY/FDKP powder(0.3 mg PYY) by pulmonary insufflation; 20% PYY/FDKP powder (0.6 mg PYY)by pulmonary insufflation. For each group food consumption was measured30 minutes after dosing, 1 hour after dosing, 2 hours after dosing, and4 hours after dosing. Data are plotted mean±SD.

FIG. 15 depicts the blood glucose concentration in female ZDF ratsadministered PYY/FDKP powder by pulmonary insufflation versusintravenously administered PYY at various times following doseadministration.

FIG. 16 depicts mean plasma concentrations of PYY in female ZDF ratsreceiving PYY/FDKP powder by pulmonary insufflation versus intravenouslyadministered PYY. The squares represent the response followingintravenous administration of PYY alone (0.6 mg). The circles representthe response following liquid instillation of PYY alone (1 mg). The downtriangles represent the response following pulmonary insufflation of 20%PYY/FDKP powder (0.6 mg PYY). The up triangles represent the responsefollowing pulmonary insufflation of 10% PYY/FDKP powder (0.3 mg PYY).The left-pointing triangles represent the response following pulmonaryinsufflation of air alone. Data are plotted as ±SD.

FIG. 17 depicts the relative drug exposure and relative bioeffect of thepresent formulations administered by pulmonary inhalation and containinginsulin, exendin, oxyntomodulin or PYY compared to subcutaneous andintravenous administration.

FIG. 18 depicts mean GLP-1 plasma levels in patients administeredvarious inhaled GLP-1 and control formulations.

FIG. 19 depicts plasma insulin levels in patients administered variousinhaled GLP-1 and control formulations.

FIG. 20 depicts gastric emptying in response to an inhaled GLP-1formulation in patients administered various inhaled GLP-1 and controlformulations.

DEFINITION OF TERMS

Prior to setting forth the invention, it may be helpful to provide anunderstanding of certain terms that will be used hereinafter:

Active Agents: As used herein “active agent” refers to drugs,pharmaceutical substances and bioactive agents. Active agents can beorganic macromolecules including nucleic acids, synthetic organiccompounds, polypeptides, peptides, proteins, polysaccharides and othersugars, fatty acids, and lipids. Peptides, proteins, and polypeptidesare all chains of amino acids linked by peptide bonds. Peptides aregenerally considered to be less than 30 amino acid residues, but mayinclude more. Proteins are polymers that can contain more than 30 aminoacid residues. The term polypeptide as is know in the art and as usedherein, can refer to a peptide, a protein, or any other chain of aminoacids of any length containing multiple peptide bonds, though generallycontaining at least 10 amino acids. The active agents can fall under avariety of biological activity classes, such as vasoactive agents,neuroactive agents, hormones, anticoagulants, immunomodulating agents,cytotoxic agents, antibiotics, antiviral agents, antigens, andantibodies. More particularly, active agents may include, in anon-limiting manner, insulin and analogs thereof, growth hormone,parathyroid hormone (PTH), ghrelin, granulocyte macrophage colonystimulating factor (GM-CSF), glucagon-like peptide 1 (GLP-1), Texas Red,alkynes, cyclosporins, clopidogrel and PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), antibodies andfragments thereof, including, but not limited to, humanized or chimericantibodies; F(ab), F(ab)₂, or single-chain antibody alone or fused toother polypeptides; therapeutic or diagnostic monoclonal antibodies tocancer antigens, cytokines, infectious agents, inflammatory mediators,hormones, and cell surface antigens. In some instances, the terms “drug”and “active agent” are used interchangeably.

Diketopiperazine: As used herein, “diketopiperazine” or “DKP” includesdiketopiperazines and salts, derivatives, analogs and modificationsthereof falling within the scope of the general Formula 1, wherein thering atoms E₁ and E₂ at positions 1 and 4 are either O or N and at leastone of the side-chains R₁ and R₂ located at positions 3 and 6respectively contains a carboxylic acid (carboxylate) group. Compoundsaccording to Formula 1 include, without limitation, diketopiperazines,diketomorpholines and diketodioxanes and their substitution analogs.

Diketopiperazines, in addition to making aerodynamically suitablemicroparticles, also facilitate the delivery of drugs by speedingabsorption into the circulation. Diketopiperazines can be formed intoparticles that incorporate a drug or particles onto which a drug can beadsorbed. The combination of a drug and a diketopiperazine can impartimproved drug stability. These particles can be administered by variousroutes of administration. As dry powders these particles can bedelivered by inhalation to specific areas of the respiratory system,depending on particle size. Additionally, the particles can be madesmall enough for incorporation into an intravenous suspension dosageform. Oral delivery is also possible with the particles incorporatedinto a suspension, tablets or capsules. Diketopiperazines may alsofacilitate absorption of an associated drug.

In one embodiment, the diketopiperazine is3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine (fumaryldiketopiperazine, FDKP). The FDKP can comprise microparticles in itsacid form or salt forms which can be aerosolized or administered in asuspension.

In another embodiment, the DKP is a derivative of3,6-di(4-aminobutyl)-2,5-diketopiperazine, which can be formed by(thermal) condensation of the amino acid lysine. Exemplary derivativesinclude 3,6-di(succinyl-4-aminobutyl)-, 3,6-di(maleyl-4-aminobutyl)-,3,6-di(glutaryl-4-aminobutyl)-, 3,6-di(malonyl-4-aminobutyl)-,3,6-di(oxalyl-4-aminobutyl)-, and3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine. The use of DKPs fordrug delivery is known in the art (see for example U.S. Pat. Nos.5,352,461, 5,503,852, 6,071,497, and 6,331,318”, each of which isincorporated herein by reference for all that it teaches regardingdiketopiperazines and diketopiperazine-mediated drug delivery). The useof DKP salts is described in co-pending U.S. patent application Ser. No.11/210,710 filed Aug. 23, 2005, which is hereby incorporated byreference for all it teaches regarding diketopiperazine salts. Pulmonarydrug delivery using DKP microparticles is disclosed in U.S. Pat. No.6,428,771, which is hereby incorporated by reference in its entirety.Further details related to adsorption of active agents onto crystallineDKP particles can be found in co-pending U.S. patent application Ser.Nos. 11/532,063 and 11/532,065 which are hereby incorporated byreference in their entirety.

Drug delivery system: As used herein, “drug delivery system” refers to asystem for delivering one or more active agents.

Dry powder: As used herein, “dry powder” refers to a fine particulatecomposition that is not suspended or dissolved in a propellant, carrier,or other liquid. It is not meant to necessarily imply a complete absenceof all water molecules.

Early phase: As used herein, “early phase” refers to the rapid rise inblood insulin concentration induced in response to a meal. This earlyrise in insulin in response to a meal is sometimes referred to asfirst-phase. In more recent sources, first-phase is sometimes used torefer to the more rapid rise in blood insulin concentration of thekinetic profile achievable with a bolus IV injection of glucose indistinction to the meal-related response.

Endocrine disease: The endocrine system is an information signal systemthat releases hormones from the glands to provide specific chemicalmessengers which regulate many and varied functions of an organism,e.g., mood, growth and development, tissue function, and metabolism, aswell as sending messages and acting on them. Diseases of the endocrinesystem include, but are not limited to diabetes mellitus, thyroiddisease, and obesity. Endocrine disease is characterized by dysregulatedhormone release (a productive pituitary adenoma), inappropriate responseto signalling (hypothyroidism), lack or destruction of a gland (diabetesmellitus type 1, diminished erythropoiesis in chronic renal failure),reduced responsiveness to signaling (insulin resistance of diabetesmellitus type 2) or structural enlargement in a critical site such asthe neck (toxic multinodular goiter). Hypofunction of endocrine glandscan occur as result of loss of reserve, hyposecretion, agenesis,atrophy, or active destruction. Hyperfunction can occur as result ofhypersecretion, loss of suppression, hyperplastic, or neoplastic change,or hyperstimulation. The term endocrine disorder encompasses metabolicdisorders.

Excursion: As used herein, “excursion” can refer to blood glucoseconcentrations that fall either above or below a pre-meal baseline orother starting point. Excursions are generally expressed as the areaunder the curve (AUC) of a plot of blood glucose over time. AUC can beexpressed in a variety of ways. In some instances there will be both afall below and rise above baseline creating a positive and negativearea. Some calculations will subtract the negative AUC from thepositive, while others will add their absolute values. The positive andnegative AUCs can also be considered separately. More sophisticatedstatistical evaluations can also be used. In some instances it can alsorefer to blood glucose concentrations that rise or fall outside a normalrange. A normal blood glucose concentration is usually between 70 and110 mg/dL from a fasting individual, less than 120 mg/dL two hours aftereating a meal, and less than 180 mg/dL after eating. While excursion hasbeen described herein in terms of blood glucose, in other contexts theterm may be similarly applied to other analytes.

Glucose elimination rate: As used herein, “glucose elimination rate” isthe rate at which glucose disappears from the blood. It is commonlydetermined by the amount of glucose infusion required to maintain stableblood glucose, often around 120 mg/dL during the study period. Thisglucose elimination rate is equal to the glucose infusion rate,abbreviated as GIR.

Hyperglycemia: As used herein, “hyperglycemia” is a higher than normalfasting blood glucose concentration, usually 126 mg/dL or higher. Insome studies hyperglycemic episodes were defined as blood glucoseconcentrations exceeding 280 mg/dL (15.6 mM).

Hypoglycemia: As used herein, “hypoglycemia” is a lower than normalblood glucose concentration, usually less than 63 mg/dL 3.5 mM),Clinically relevant hypoglycemia is defined as blood glucoseconcentration below 63 mg/dL or causing patient symptoms such ashypotonia, flush and weakness that are recognized symptoms ofhypoglycemia and that disappear with appropriate caloric intake. Severehypoglycemia is defined as a hypoglycemic episode that required glucagoninjections, glucose infusions, or help by another party.

In proximity: As used herein, “in proximity,” as used in relation to ameal, refers to a period near in time to the beginning of a meal orsnack.

Microparticles: As used herein, the term “microparticles” includesparticles of generally 0.5 to 100 microns in diameter and particularlythose less than 10 microns in diameter. Various embodiments will entailmore specific size ranges. The microparticles can be assemblages ofcrystalline plates with irregular surfaces and internal voids as istypical of those made by pH controlled precipitation of the DKP acids.In such embodiments the active agents can be entrapped by theprecipitation process or coated onto the crystalline surfaces of themicroparticle. The microparticles can also be spherical shells orcollapsed spherical shells comprised of DKP salts with the active agentdispersed throughout. Typically such particles can be obtained by spraydrying a co-solution of the DKP and the active agent. The DKP salt insuch particles can be amorphous. The forgoing descriptions should beunderstood as exemplary. Other forms of microparticles are contemplatedand encompassed by the term.

Obesity: is a condition in which excess body fat has accumulated to suchan extent that health may be negatively affected. Obesity is typicallyassessed by BMI (body mass index) with BMI of greater than 30 kg/m².

Peripheral tissue: As used herein, “peripheral tissue” refers to anyconnective or interstitial tissue that is associated with an organ orvessel.

Periprandial: As used herein, “periprandial” refers to a period of timestarting shortly before and ending shortly after the ingestion of a mealor snack.

Postprandial: As used herein, “postprandial” refers to a period of timeafter ingestion of a meal or snack. As used herein, late postprandialrefers to a period of time 3, 4, or more hours after ingestion of a mealor snack.

Potentiation: Generally, potentiation refers to a condition or actionthat increases the effectiveness or activity of some agent over thelevel that the agent would otherwise attain. Similarly it may referdirectly to the increased effect or activity. As used herein,“potentiation” particularly refers to the ability of elevated bloodinsulin concentrations to boost effectiveness of subsequent insulinlevels to, for example, raise the glucose elimination rate.

Prandial: As used herein, “prandial” refers to a meal or a snack.

Preprandial: As used herein, “preprandial” refers to a period of timebefore ingestion of a meal or snack.

Pulmonary inhalation: As used herein, “pulmonary inhalation” is used torefer to administration of pharmaceutical preparations by inhalation sothat they reach the lungs and in particular embodiments the alveolarregions of the lung. Typically inhalation is through the mouth, but inalternative embodiments in can entail inhalation through the nose.

Reduction in side effects: As used herein, the term “reduction” whenused with regard to side effects, refers to a lessening of the severityof one or more side effects noticeable to the patient or a healthcareworker whose care they are under, or the amelioration of one or moreside effects such that the side effects are no longer debilitating or nolonger noticeable to the patient.

Side Effects: As used herein, the term “side effects” refers tounintended, and undesirable, consequences arising from active agenttherapy. In a non-limiting example, common side effects of GLP-1include, but are not limited to, nausea, vomiting and profuse sweating.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a composition, when administeredto a human or non-human patient, to provide a therapeutic benefit suchas an amelioration of symptoms, e.g., an amount effective to stimulatethe secretion of endogenous insulin. In certain circumstances a patientsuffering from a disorder may not present symptoms of being affected.Thus a therapeutically effective amount of a composition is also anamount sufficient to prevent the onset of symptoms of a disease.

DETAILED DESCRIPTION

Glucagon-like peptide 1 (GLP-1) has been studied as a treatment forhyperglycemia associated with Type 2 diabetes mellitus by various routesof administration. GLP-1 as disclosed in the literature is a 30 or 31amino acid incretin hormone, released from the intestinal endocrineL-cells in response to eating fat, carbohydrates, and proteins. GLP-1 isproduced as a result of proteolytic cleavage of proglucagon and theactive form is identified as GLP-1(7-36) amide. Secretion of thispeptide hormone is found to be impaired in individuals with type 2diabetes mellitus making this peptide hormone a primary candidate forpotential treatments of this and other related diseases.

In the non-disease state, GLP-1 is secreted from intestinal L-cells inresponse to orally ingested nutrients, particularly sugars. GLP-1 haseffects on the gastrointestinal tract (GI) and brain includingstimulating meal-induced insulin release from the pancreas. The GLP-1effect in the pancreas is glucose dependent so the risk of GLP-1 inducedhypoglycemia is minimal when the hormone is administered exogenously.GLP-1 also promotes all steps in insulin biosynthesis and directlystimulates β-cell growth, survival, and differentiation. The combinationof these effects results in increased β-cell mass. Furthermore, GLP-1receptor signaling results in a reduction of β-cell apoptosis andfurther contributes to increased β-cell mass.

In the gastrointestinal tract, GLP-1 as reported in the literatureinhibits motility, increases the insulin secretion in response toglucose, and decreases the glucagon secretion. These effects combine toreduce postprandial glucose excursions. Experiments in rodents in whichGLP-1 was given by central administration (intracerebroventricular oricv) have shown GLP-1 to inhibit food intake, suggesting thatperipherally released GLP-1 can enter the systemic circulation and mayhave its effect on the brain. This effect may be the result ofcirculating GLP-1 accessing GLP-1 receptors in the brain subformicalorgan and area postrema. These areas of the brain are known to beinvolved in the regulation of appetite and energy homeostasis.Interestingly, gastric distension activates GLP-1 containing neurons inthe caudal nucleus of the solitary tract, predicting a role forcentrally expressed GLP-1 as an appetite suppressant. These hypothesesare supported by studies employing the GLP-1 receptor antagonist,exendin(9-39), where opposite effects were seen. In humans, administeredGLP-1 has a satiating effect, and when given by continuous subcutaneousinfusion over a 6 weeks regime, patients with diabetes exhibited areduction in appetite leading to significant reductions in body weight.

GLP-1 has also been shown to increase insulin secretion and normalizeboth fasting and postprandial blood glucose when given as a continuousintravenous infusion to patients with type 2 diabetes. In addition,GLP-1 infusion has been shown to lower glucose levels in patientspreviously treated with non-insulin oral medication and in patientsrequiring insulin therapy after failure on sulfonylurea therapy.However, the effects of a single subcutaneous injection of GLP-1provided disappointing results, as is noted in the art and discussedherein below. Although high plasma levels of immunoreactive GLP-1 wereachieved, insulin secretion rapidly returned to pretreatment values andblood glucose concentrations were not normalized. Repeated subcutaneousadministrations were required to achieve fasting blood glucoseconcentrations comparable to those observed with intravenousadministration. Continuous subcutaneous administration of GLP-1 for 6weeks was shown to reduce fasting and postprandial glucoseconcentrations and lower HbAlc levels. The short-lived effectiveness ofsingle subcutaneous injections of GLP-1 is related to its circulatoryinstability. GLP-1 is metabolized in plasma in vitro by dipeptidylpeptidase-IV (DPP-IV). GLP-1 is rapidly degraded by DPP-IV by theremoval of amino acids 7 and 8 from the N-terminus. The degradationproduct, GLP-1(9-36) amide, is not active. DPP-4 circulates within theblood vessels and is membrane bound in the vasculature of thegastrointestinal tract and kidney and has been identified on lymphocytesin the lung.

The utility of GLP-1, and GLP-1 analogs, as a treatment forhyperglycemia associated with Type 2 diabetes mellitus has been studiedfor over 20 years. Clinically, GLP-1 reduces blood glucose, postprandialglucose excursions and food intake. It also increases satiety. Takentogether, these actions define the unique and highly desirable profileof an anti-diabetic agent with the potential to promote weight loss.Despite these advantages, the utility of GLP-1 as a diabetes treatmentis hindered because it requires administration by injection and GLP-1has a very short circulating half-life because it is rapidly inactivatedby the enzyme dipeptidyl peptidase (DPP)-IV. Thus to achievetherapeutically effective concentrations of GLP-1, higher GLP-1 dosesare required. However, based on extensive literature evaluation, whenactive GLP-1 concentrations exceed 100 pmol/L in blood plasma, acombination of side effects/adverse effects are typically observed,including profuse sweating, nausea, and vomiting.

To address the challenge of GLP-1's limited half-life, severallong-acting GLP-1 analogs have been or are currently in development.Long-acting GLP-1 analogs including liraglutide (Novo Nordisk,Copenhagen, Denmark), exenatide (exendin-4; Byetta®) (Amylin Inc., SanDiego, Calif.), and exenatide-LAR (Eli Lilly, Indianapolis, Ind.)) thatare resistant to degradation are called “incretin mimetics,” and havebeen investigated in clinical trials. Exenatide is an approved therapyfor type 2 diabetes. These products are formulations for subcutaneousadministration, and these formulations are known to have significantlimitations due to degradation in peripheral tissue, vascular tissueand/or the liver. For example, exenatide (Byetta®, AmylinPharmaceuticals), a compound with approximately 50% amino acid homologywith GLP-1, has a longer circulating half-life than GLP-1. This producthas been approved by the Food and Drug Administration for the treatmentof hyperglycemia associated with Type 2 diabetes mellitus. While thecirculating half-life of exenatide is longer than that of GLP-1, it isstill requires patients to inject the drug twice daily. Exenatidetherapy is further complicated by a poor side effect profile including asignificant incidence of nausea. Additionally, while this long-actingtherapeutic approach may provide patient convenience and facilitatecompliance, the pharmacokinetic profiles for long-acting GLP-1 analogsadministered by injection can be radically different from those ofendogenously secreted hormones. This regimen may be effective, but doesnot mimic normal physiology.

While the current approaches/advances to treating diabetes and/orhyperglycemia using long-acting GLP-1 analogs administered bysubcutaneous injections have been able to provide acceptable treatmentfor diabetes, the treatments do not mimic the body's natural physiology.For example, in healthy individuals, endogenous GLP-1 is secreted onlyafter a meal and only in short bursts as needed. By contrast,long-acting GLP-1 analogs provide drug exposure for time periodsexceeding the postprandial phase. Thus, the ideal GLP-1 therapy might beone in which the drug is administered at mealtime with exposure limitedto the postprandial period. The pulmonary route of drug administrationhas the potential to provide such a treatment, but, to our knowledge,has not been previously explored due to the presence of DPP-IV in thelungs.

An alternative approach to prolonging the circulating half-life of GLP-1involves the development of DPP-IV inhibitors because DPP-IV is theenzyme responsible for GLP-1 metabolism. Inhibition of DPP-IV has beenshown to increase the half-life of endogenous GLP-1. Dipeptidylpeptidase IV inhibitors include vildagliptin (Galvus®) developed byNovartis (Basel, Switzerland) and Januvia® (sitagliptin) developed byMerck (Whitehouse Station, N.J.).

In contrast to healthy individuals, the current methods to treatpatients with hyperglycemia and type 2 diabetes use long acting GLP-1analogs and DPP-IV inhibitors which provide drug exposure for timeperiods exceeding the postprandial phase. Accordingly, these currentmethods are not devoid of detrimental or negative side effects such asprofuse sweating, nausea and vomiting, which impact on the patient'squality of life. Therefore, the inventors have identified the need todevelop new methods of treatment of diseases using a drug deliverysystem which increases pharmacodynamic response to the drug at lowersystemic exposure, while avoiding unwanted side effects. Additionally,the inventors identified the need to deliver drugs directly to thearterial circulation using a noninvasive method.

In embodiments herein, there is disclosed a method for the treatment ofdisease, including, endocrine disease, such as diabetes, hyperglycemiaand obesity. The inventors have identified the need to deliver drugsdirectly to the systemic circulation, in particular, the arterialcirculation in a noninvasive fashion so that the drug reaches the targetorgan(s) prior to returning through the venous system. This approach mayparadoxically result in a higher peak target organ exposure to activeagents than would result from a comparable administration via anintravenous, subcutaneous or other parenteral route. A similar advantagecan be obtained versus oral administration as, even with formulationsproviding protection from degradation in digestive tract, uponabsorption the active agent will enter the venous circulation.

In one embodiment, the drug delivery system can be used with any type ofactive agent that is rapidly metabolized and/or degraded by directcontact with the local degradative enzymes or other degradativemechanisms, for example oxidation, phosphorylation or any modificationof the protein or peptide, in the peripheral or vascular venous tissueencountered with other routes of administration such as oral,intravenous, transdermal, and subcutaneous administration. In thisembodiment, the method can comprise the step of identifying andselecting an active agent which activity is metabolized or degraded byoral, subcutaneous or intravenous administration. For example, due toliability, subcutaneous injection of GLP-1 has not led to effectivelevels of GLP-1 in the blood. This contrasts with peptides such asinsulin which can be delivered effectively by such modes ofadministration.

In certain embodiments, the method of treatment of a disease or disordercomprises the step of selecting a suitable carrier for inhalation anddelivering an active substance to pulmonary alveoli. In this embodiment,the carrier can be associated with one or more active agents to form adrug/carrier complex which can be administered as a composition thatavoids rapid degradation of the active agent in the peripheral andvascular venous tissue of the lung. In one embodiment, the carrier is adiketopiperazine.

The method described herein can be utilized to deliver many types ofactive agents, including biologicals. In particular embodiments, themethod utilizes a drug delivery system that effectively delivers atherapeutic amount of an active agent, including peptide hormones,rapidly into the arterial circulation. In one embodiment, the one ormore active agents include, but are not limited to peptides such asglucagon-like peptide 1 (GLP-1), proteins, lipokines, small moleculepharmaceuticals, nucleic acids and the like, which is/are sensitive todegradation or deactivation; formulating the active agent into a drypowder composition comprising a diketopiperazine and delivering theactive agent(s) into the systemic circulation by pulmonary inhalationusing a cartridge and a dry powder inhaler. In one embodiment, themethod comprises selecting a peptide that is sensitive to enzymes in thelocal vascular or peripheral tissue of, for example, the dermis andlungs. The present method allows the active agent to avoid or reducecontact with peripheral tissue, venous or liver metabolism/degradation.

In another embodiment, for systemic delivery the active agent should nothave specific receptors in the lungs.

In alternate embodiments, the drug delivery system can also be used todeliver therapeutic peptides or proteins of naturally occurring,recombinant, or synthetic origin for treating disorders or diseases,including, but not limited to adiponectin, cholecystokinin (CCK),secretin, gastrin, glucagon, motilin, somatostatin, brain natriureticpeptide (BNP), atrial natriuretic peptide (ANP), parathyroid hormone,parathyroid hormone related peptide (PTHrP), IGF-1, growth hormonereleasing factor (GHRF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), anti-IL-8 antibodies, IL-8 antagonists includingABX-IL-8; integrin beta-4 precursor (ITB4) receptor antagonist,enkephalins, nociceptin, nocistatin, orphanin FQ2, calcitonin, CGRP,angiotensin, substance P, neurokinin A, pancreatic polypeptide,neuropeptide Y, delta-sleep-inducing peptide, prostaglandings includingPG-12, LTB receptor blockers including, LY29311, BIIL 284, CP105696;vasoactive intestinal peptide; triptans such as sumatriptan andlipokines such as C16:1n7 or palmitoleate. In yet another embodiment,the active agent is a small molecule drug.

In one embodiment, the method of treatment is directed to the treatmentof diabetes, hyperglycemia and/or obesity using, for example,formulations comprising glucagon-like peptide 1 (GLP-1), oxyntomodulin(OXN), or peptide YY(3-36) (PYY) either alone or in combination with oneanother, or in combination with one or more active agents.

In an exemplary embodiment, a method for treating obesity, diabetesand/or hyperglycemia comprising administering to a patient in need oftreatment a dry powder composition or formulation comprising GLP-1,which stimulates the rapid secretion of endogenous insulin frompancreatic β-cells without causing unwanted side effects such as profusesweating, nausea, and vomiting. In one embodiment, the method oftreating disease can be applied to a patient, including a mammal,suffering with obesity, Type 2 diabetes mellitus and/or hyperglycemia atdosages ranging from about 0.02 to about 3 mg of GLP-1 in theformulation in a single dose. The method of treating hyperglycemia,diabetes, and/or obesity can be designed so that the patient can receiveat least one dose of a GLP-1 formulation in proximity to a meal orsnack. In this embodiment, the dose of GLP-1 can be selected dependingon the patient's requirements. In one embodiment, pulmonaryadministration of GLP-1 can comprise a GLP-1 dose greater than 3 mg forexample, in treating patients with type 2 diabetes.

In embodiments of the invention, the GLP-1 formulation is administeredby inhalation such as by pulmonary administration. In this embodiment,pulmonary administration can be accomplished by providing the GLP-1 in adry powder formulation for inhalation. The dry powder formulation is astable composition and can comprise microparticles which are suitablefor inhalation and which dissolve rapidly in the lung and rapidlydeliver GLP-1 to the pulmonary circulation. Suitable particle sizes forpulmonary administration can be less than 10 μm in diameter, andpreferably less than 5 μm. Exemplary particle sizes that can reach thepulmonary alveoli range from about 0.5 μm to about 5.8 μm in diameter.Such sizes refer particularly to aerodynamic diameter, but often alsocorrespond to actually physical diameter as well. Such particles canreach the pulmonary capillaries, and can avoid extensive contact withthe peripheral tissue in the lung. In this embodiment, the drug can bedelivered to the arterial circulation in a rapid manner and avoiddegradation of the active ingredient by enzymes or other mechanismsprior to reaching its target or site of action in the body. In oneembodiment, dry powder compositions for pulmonary inhalation comprisingGLP-1 and FDKP can comprise microparticles wherein from about 35% toabout 75% of the microparticles have an aerodynamic diameter of lessthan 5.8 μm.

In one embodiment, the dry powder formulation for use with the methodscomprises particles comprising a GLP-1 molecule and a diketopiperazineor a pharmaceutically acceptable salt thereof. In this and otherembodiments, the dry powder composition of the present inventioncomprises one or more GLP-1 molecules selected from the group consistingof a native GLP-1, a GLP-1 metabolite, a long acting GLP-1, a GLP-1derivative, a GLP-1 mimetic, an exendin, or an analog thereof. GLP-1analogs include, but are not limited to GLP-1 fusion proteins, such asalbumin linked to GLP-1

In an exemplary embodiment, the method comprises the administration ofthe peptide hormone GLP-1 to a patient for the treatment ofhyperglycemia and/or diabetes, and obesity. The method comprisesadministering to a patient in need of treatment an effective amount ofan inhalable composition or formulation comprising a dry powderformulation comprising GLP-1 which stimulates the rapid secretion ofendogenous insulin from pancreatic β-cells without causing unwanted sideeffects, including, profuse sweating, nausea, and vomiting. In oneembodiment, the method of treating disease can be applied to a patient,including a mammal, suffering with Type 2 diabetes mellitus and/orhyperglycemia at dosages ranging from about 0.01 mg to about 3 mg, orfrom about 0.2 mg to about 2 mg of GLP-1 in the dry powder formulation.In one embodiment, the patient or subject to be treated is a human. TheGLP-1 can be administered immediately before a meal (preprandially), atmealtime (prandially), and/or at about 15, 30 or 45 minutes after a meal(postprandially). In one embodiment, a single dose of GLP-1 can beadministered immediately before a meal and another dose can beadministered after a meal. In a particular embodiment, about 0.5 mg toabout 1.5 mg of GLP-1 can be administered immediately before a meal,followed by 0.5 mg to about 1.5 mg about 30 minutes after a meal. Inthis embodiment, the GLP-1 can be formulated with inhalation particlessuch as a diketopiperazines with or without pharmaceutical carriers andexcipients. In one embodiment, pulmonary administration of the GLP-1formulation can provide plasma concentrations of GLP-1 greater than 100pmol/L without inducing unwanted adverse side effects, such as profusesweating, nausea and vomiting to the patient.

In another embodiment, a method for treating a patient including a humanwith type 2 diabetes and hyperglycemia is provided, the method comprisesadministering to the patient an inhalable GLP-1 formulation comprisingGLP-1 in a concentration of from about 0.5 mg to about 3 mg in FDKPmicroparticles wherein the levels of glucose in the blood of the patientare reduced to fasting plasma glucose concentrations of from 85 to 70mg/dL within about 20 min after dosing without inducing nausea orvomiting in the patient. In one embodiment, pulmonary administration ofGLP-1 at concentration greater than 0.5 mg in a formulation comprisingFDKP microparticles lacks inhibition of gastric emptying.

In one embodiment, GLP-1 can be administered either alone as the activeingredient in the composition, or with a dipeptidyl peptidase (DPP-IV)inhibitor such as sitagliptin or vildagliptin, or with one or more otheractive agents. DPP-IV is a ubiquitously expressed serine protease thatexhibits postproline or alanine peptidase activity, thereby generatingbiologically inactive peptides via cleavage at the N-terminal regionafter X-proline or X-alanine, wherein X refers to any amino acid.Because both GLP-1 and GIP (glucose-dependent insulinotropic peptide)have an alanine residue at position 2, they are substrates for DPP-IV.DPP-IV inhibitors are orally administered drugs that improve glycemiccontrol by preventing the rapid degradation of incretin hormones,thereby resulting in postprandial increases in levels of biologicallyactive intact GLP-1 and GIP.

In this embodiment, the action of GLP-1 can be further prolonged oraugmented in vivo if required, using DPP-IV inhibitors. The combinationof GLP-1 and DPP-IV inhibitor therapy for the treatment of hyperglycemiaand/or diabetes allows for reduction in the amount of active GLP-1 thatmay be needed to induce an appropriate insulin response from the β-cellsin the patient. In another embodiment, the GLP-1 can be combined, forexample, with other molecules other than a peptide, such as, forexample, metformin. In one embodiment, the DPP-IV inhibitor or othermolecules, including metformin, can be administered by inhalation in adry powder formulation together with the GLP-1 in a co-formulation, orseparately in its own dry powder formulation which can be administeredconcurrently with or prior to GLP-1 administration. In one embodiment,the DPP-IV inhibitor or other molecules, including metformin, can beadministered by other routes of administration, including orally. In oneembodiment, the DPP-IV inhibitor can be administered to the patient indoses ranging from about 1 mg to about 100 mg depending on the patient'sneed. Smaller concentration of the DPP-IV inhibitor may be used whenco-administered, or co-formulated with GLP-1. In this embodiment, theefficacy of GLP-1 therapy may be improved at reduced dosage ranges whencompared to current dosage forms.

In embodiments described herein, GLP-1 can be administered at mealtime(in proximity in time to a meal or snack). In this embodiment, GLP-1exposure can be limited to the postprandial period so it does not causethe long acting effects of current therapies. In embodiments wherein theDPP-IV inhibitor is co-administered, the DPP-IV inhibitor may be givento the patient prior to GLP-1 administration at mealtime. The amounts ofDPP-IV inhibitor to be administered can range, for example, from about0.10 mg to about 100 mg, depending on the route of administrationselected. In further embodiment, one or more doses of the GLP-1 can beadministered after the beginning of the meal instead of, or in additionto, a dose administered in proximity to the beginning of a meal orsnack. For example, one or more doses can be administered 15 to 120minutes after the beginning of a meal, such as at 30, 45, 60, or 90minutes.

In one embodiment, the drug delivery system can be utilized in a methodfor treating obesity so as to control or reduce food consumption in ananimal such as a mammal. In this embodiment, patients in need oftreatment or suffering with obesity are administered a therapeuticallyeffective amount of an inhalable composition or formulation comprisingGLP-1, an exendin, oxyntomodulin, peptide YY(3-36), or combinationsthereof, or analogs thereof, with or without additional appetitesuppressants known in the art. In this embodiment, the method istargeted to reduce food consumption, inhibit food intake in the patient,decrease or suppress appetite, and/or control body weight.

In one embodiment, the inhalable formulation comprises a dry powderformulation comprising the above-mentioned active ingredient with adiketopiperazine, including 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine;wherein X is selected from the group consisting of succinyl, glutaryl,maleyl, and fumaryl, or a salt of the diketopiperazine. In thisembodiment, the inhalable formulation can comprise microparticles forinhalation comprising the active ingredient with the aerodynamiccharacteristics as described above. In one embodiment, the amount ofactive ingredient can be determined by one of ordinary skill in the art,however, the present microparticles can be loaded with various amountsof active ingredient as needed by the patient. For example, foroxyntomodulin, the microparticles can comprise from about 1% (w/w) toabout 75% (w/w) of the active ingredient in the formulation. In certainembodiments, the inhalable formulations can comprise from about 10%(w/w) to about 30% (w/w) of the pharmaceutical composition and can alsocomprise a pharmaceutically acceptable carrier, or excipient, such as asurfactant, such as polysorbate 80. In this embodiment, oxyntomodulincan be administered to the patient from once to about four times a dayor as needed by the patient with doses ranging from about 0.05 mg up toabout 5 mg in the formulation. In particular embodiments, the dosage tobe administered to a subject can range from about 0.1 mg to about 3.0 mgof oxyntomodulin. In one embodiment, the inhalable formulation cancomprise from about 50 pmol to about 700 pmol of oxyntomodulin in theformulation.

In embodiments disclosed herein wherein PYY is used as the activeingredient, a dry powder formulation for pulmonary delivery can be madecomprising from about 0.10 mg to about 3.0 mg of PYY per dose. Incertain embodiments, the formulation can comprise a dry powdercomprising PYY in an amount ranging from about 1% to about 75% (w/w) ofthe peptide in the formulation. In particular embodiments, the amount ofPYY in the formulation can be 5%, 10%, 15%, or 20% (w/w) and furthercomprising a diketopiperazine. In one embodiment, the PYY isadministered in a formulation comprising a diketopiperazine, such asFDKP or a salt thereof, including sodium salts. In certain embodiments,PYY can be administered to a subject in dosage forms so that plasmaconcentrations of PYY after administration are from about 4 pmol/L toabout 100 pmol/L or from about 10 pmol/L to about 50 pmol/L. In anotherembodiment, the amount of PYY can be administered, for example, inamounts ranging from about 0.01 mg to about 30 mg, or from about 5 mg toabout 25 mg in the formulation. Other amounts of PYY can be determinedas described, for example, in Savage et al. Gut 1987 February;28(2):166-70; which disclosure is incorporated by reference herein. ThePYY and/or analog, or oxyntomodulin and/or analog formulation can beadministered preprandially, prandially, periprandially, orpostprandially to a subject, or as needed and depending on the patientphysiological condition.

In one embodiment, the formulation comprising the active ingredient canbe administered to the patient in a dry powder formulation by inhalationusing a dry powder inhaler such as the inhaler disclosed, for example,in U.S. Pat. No. 7,305,986 and U.S. patent application Ser. No.10/655,153 (US 2004/0182387), which disclosures are incorporated hereinby reference. Repeat inhalation of dry powder formulation comprising theactive ingredient can also be administered between meals and daily asneeded. In some embodiments, the formulation can be administered once,twice, three or four times a day.

In still yet a further embodiment, the method of treating hyperglycemiaand/or diabetes comprises the administration of an inhalable dry powdercomposition comprising a diketopiperazine having the formula2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl. In thisembodiment, the dry powder composition can comprise a diketopiperazinesalt. In still yet another embodiment of the present invention, there isprovided a dry powder composition, wherein the diketopiperazine is2,5-diketo-3,6-di-(4-fumaryl-aminobutyl)piperazine, with or without apharmaceutically acceptable carrier, or excipient.

In certain embodiments, the method of treatment can comprise a drypowder formulation for inhalation comprising GLP-1, wherein the GLP-1molecule is native GLP-1, or an amidated GLP-1 molecule, wherein theamidated GLP-1 molecule is GLP-1(7 36) amide, or combinations thereof.In one embodiment, the GLP-1 can be an analog such as exenatide.

In one embodiment, a patient is administered an inhalable GLP-1formulation in a dosing range wherein the amount of GLP-1 is from about0.01 mg to about 3 mg, or from about 0.02 mg to about 2.5 mg, or fromabout 0.2 mg to about 2 mg of the formulation. In one embodiment, apatient with type 2 diabetes can be given a GLP-1 dose greater than 3mg. In this embodiment, the GLP-1 can be formulated with inhalationparticles such as a diketopiperazines with or without pharmaceuticalcarriers and excipients. In one embodiment, pulmonary administration ofthe GLP-1 formulation can provide plasma concentrations of GLP-1 greaterthan 100 pmol/L without inducing unwanted adverse side effects, such asprofuse sweating, nausea and vomiting to the patient.

In another embodiment, GLP-1 can be administered with insulin as acombination therapy and given prandially for the treatment ofhyperglycemia and/or diabetes, for example, Type 2 diabetes mellitus. Inthis embodiment, GLP-1 and insulin can be co-formulated in a dry powderformulation or administered separately to a patient in their ownformulation. In one embodiment, wherein the GLP-1 and insulin areco-administered, both active ingredients can be co-formulated, forexample, the GLP-1 and insulin can be prepared in a dry powderformulation for inhalation using a diketopiperazine particle asdescribed above. Alternatively, the GLP-1 and insulin can be formulatedseparately, wherein each formulation is for inhalation and comprise adiketopiperazine particle. In one embodiment the GLP-1 and the insulinformulations can be admixed together in their individual powder form tothe appropriate dosing prior to administration. In this embodiment, theinsulin can be short-, intermediate-, or long-acting insulin and can beadministered prandially.

In one embodiment for the treatment of Type 2 diabetes usingco-administration of GLP-1 and insulin, an inhalable formulation ofGLP-1 can be administered to a patient prandially, simultaneously, orsequentially to an inhalable formulation of insulin such asinsulin/FDKP. In this embodiment, in a Type 2 diabetic, can stimulateinsulin secretion from the patient's pancreas, which can delay diseaseprogression by preserving β-cell function (such as by promoting β-cellgrowth) while prandially-administered insulin can be used as insulinreplacement which mimics the body's normal response to a meal. Incertain embodiments of the combination therapy, the insulin formulationcan be administered by other routes of administration. In thisembodiment, the combination therapy can be effective in reducing insulinrequirements in a patient to maintain the euglycemic state. In oneembodiment, the combination therapy can be applied to patients sufferingwith obesity and/or Type 2 diabetes who have had diabetes for less than10 years and are not well controlled on diet and exercise orsecretagogues. In one embodiment, the patient population for receivingGLP-1 and insulin combination therapy can be characterized by havingβ-cell function greater than about 25% of that of a normal healthyindividual and/or, insulin resistance of less than about 8% and/or canhave normal gastric emptying. In one embodiment, the inhalable GLP-1 andinsulin combination therapy can comprise a rapid acting insulin or along acting insulin such as insulin glulisine (APIDRA®), insulin lispro(HUMALOG®) or insulin aspart (NOVOLOG®), or a long acting insulin suchas insulin detemir (LEVEMIR®) or insulin glargine (LANTUS®), which canbe administered by an inhalation powder also comprising FDKP or by otherroutes of administration.

In another embodiment, a combination therapy for treating type 2diabetes can comprise administering to a patient in need of treatment aneffective amount of an inhalable insulin formulation comprising aninsulin and a diketopiperazine, wherein the insulin can be a nativeinsulin peptide, a recombinant insulin peptide, and furtheradministering to the patient a long acting insulin analog which can beprovided by inhalation in a formulation comprising a diketopiperazine orby another route of administration such as by subcutaneous injection.The method can further comprise the step of administering to the patientan effective amount of a DPP IV inhibitor. In one embodiment, the methodcan comprise administering to a patient in need of treatment, aformulation comprising a rapid acting or long acting insulin moleculeand a diketopiperazine in combination with formulation comprising a longacting GLP-1, which can be administered separately and/or sequentially.GLP-1 therapy for treating diabetes in particular type 2 diabetes can beadvantageous since administration of GLP-1 alone in a dry powderinhalable formulation or in combination with insulin or non-insulintherapies can reduce the risk of hypoglycemia.

In another embodiment, rapid acting GLP-1 and a diketopiperazineformulation can be administered in combination with a long acting GLP-1,such as exendin, for the treatment of diabetes, which can be bothadministered by pulmonary inhalation. In this embodiment, a diabeticpatient suffering, for example, with Type 2 diabetes, can beadministered prandially an effective amount of an inhalable formulationcomprising GLP-1 so as to stimulate insulin secretion, whilesequentially or sometime after such as from mealtime up to about 45 min,thereafter administering a dose of exendin-4. Administration ofinhalable GLP-1 can prevent disease progression by preserving β-cellfunction while exendin-4 can be administered twice daily atapproximately 10 hours apart, which can provide basal levels of GLP-1that can mimic the normal physiology of the incretin system in apatient. Both rapid acting GLP-1 and a long acting GLP-1 can beadministered in separate, inhalable formulations. Alternatively, thelong acting GLP-1 can be administered by other methods of administrationincluding, for example, transdermally, intravenously or subcutaneously.

In one embodiment, prandial administration of a short-acting and longacting GLP-1 combination may result in increased insulin secretion,greater glucagon suppression and a longer delay in gastric emptyingcompared to long-acting GLP-1 administered alone. The amount of longacting GLP-1 administered can vary depending on the route ofadministration. For example, for pulmonary delivery, the long actingGLP-1 can be administered in doses from about 0.1 mg to about 1 mg peradministration, immediately before a meal or at mealtime, depending onthe form of GLP-1 administered to the patient.

In one embodiment, the present method can be applied to the treatment ofobesity. A therapeutically effective amount of an inhalable GLP-1formulation can be administered to a patient in need of treatment,wherein an inhalable dry powder, GLP-1 formulation comprises GLP-1 and adiketopiperazine as described above. In this embodiment, the inhalableGLP-1 formulation can be administered alone or in combination with oneor more endocrine hormone and/or anti-obesity active agents for thetreatment of obesity. Exemplary endocrine hormones and/or anti-obesityactive agents include, but are not limited to, peptide YY,oxyntomodulin, amylin, amylin analogs such as pramlintide acetate, andthe like. In one embodiment, the anti-obesity agents can be administeredin a co-formulation in a dry powder inhalable composition alone or incombination with GLP-1 together or in a separate inhalable dry powdercomposition for inhalation. Alternatively, in the combination of GLP-1with one or more anti-obesity agents, or agents that can cause satiety,the GLP-1 formulation can be administered in a dry powder formulationand the anti-obesity agent can be administered by alternate routes ofadministration. In this embodiment, a DPP-IV inhibitor can beadministered to enhance or stabilize GLP-1 delivery into the pulmonaryarterial circulation. In another embodiment, the DPP-IV inhibitor can beprovided in combination with an insulin formulation comprising adiketopiperazine. In this embodiment, the DPP-IV inhibitor can beformulated in a diketopiperazine for inhalation or it can beadministered in other formulation for other routes of administrationsuch as by subcutaneous injection or oral administration.

In an embodiment, a kit for treating diabetes and/or hyperglycemia isprovided which comprises a medicament cartridge for inhalationcomprising a GLP-1 formulation and an inhalation device which isconfigured to adapt or securely engage the cartridge. In thisembodiment, the kit can further comprise a DPP-IV inhibitorco-formulated with GLP-1, or in a separate formulation for inhalation ororal administration as described above. In variations of thisembodiment, the kit does not include the inhalation device which can beprovided separately.

In one embodiment, the present combination therapy using the drugdelivery system can be applied to treat metabolic disorders orsyndromes. In this embodiment, the drug delivery formulation cancomprise a formulation comprising a diketopiperazine and an activeagent, including GLP-1 and/or a long acting GLP-1 alone or incombination with one or more active agents such as a DPP-IV inhibitorand exendin, targeted to treat the metabolic syndrome. In thisembodiment, at least one of the active agents to be provided to thesubject in need of treatment and who may exhibit insulin resistance canbe administered by pulmonary inhalation.

In another embodiment, the pulmonary administration of an inhalable drypowder formulation comprising GLP-1 and a diketopiperazine can be usedas a diagnostic tool to diagnose the level or degree of progression oftype 2 diabetes in a patient afflicted with diabetes in order toidentify the particular treatment regime suitable for the patient to betreated. In this embodiment, a method for diagnosing the level ofdiabetes progression in a patient identified as having diabetes, themethod comprising administering to the patient a predetermined amount ofan inhalable dry powder formulation comprising GLP-1 and adiketopiperazine and measuring the endogenous insulin production orresponse. The administration of the inhalable dry powder formulationcomprising GLP-1 can be repeated with predetermined amounts of GLP-1until the appropriate levels of an insulin response is obtained for thatpatient to determine the required treatment regime required by thepatient. In this embodiment, if a patient insulin response isinadequate, the patient may require alternative therapies. Patients whoare sensitive or insulin-responsive can be treated with a GLP-1formulation comprising a diketopiperazine as a therapy. In this manner,the specific amount of GLP-1 can be administered to a patient in orderto achieve an appropriate insulin response to avoid hypoglycemia. Inthis and other embodiments, GLP-1 can induce a rapid release ofendogenous insulin which mimics the normal physiology of insulin releasein a patient.

In one embodiment, the present drug delivery system can be applied totreat metabolic disorders or syndromes. In this embodiment, the drugdelivery formulation can comprise a formulation comprising adiketopiperazine and an active agent, including GLP-1 and/or a longacting GLP-1 alone or in combination with one or more active agents suchas a DPP-IV inhibitor and exendin, targeted to treat the metabolicsyndrome. In this embodiment, at least one of the active agents to beprovided to the subject in need of treatment and who may exhibit insulinresistance can be administered by pulmonary inhalation.

EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples elucidate representativetechniques that function well in the practice of the present invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

Example 1 Administration of GLP-1 in an Inhalable Dry Powder to HealthyAdult Males

GLP-1 has been shown to control elevated blood glucose in humans whengiven by intravenous (iv) or subcutaneous (sc) infusions or by multiplesubcutaneous injections. Due to the extremely short half-life of thehormone, continuous subcutaneous infusion or multiple daily subcutaneousinjections would be required to achieve clinical efficacy. Neither ofthese routes is practical for prolonged clinical use. Applicants havefound in animal experiments that when GLP-1 was administered byinhalation, therapeutic levels could be achieved. The results of thesestudies can be found, for example, in U.S. patent application Ser. No.11/735,957, the disclosure of which is incorporated by reference herein.

In healthy individuals, several of the actions of GLP-1, includingreduction in gastric emptying, increased satiety, and suppression ofinappropriate glucagon secretion appear to be linked to the burst ofGLP-1 released as meals begin. By supplementing this early surge inGLP-1 with a formulation of GLP-1 and2,5-diketo-3,6-di(4-fumaryl-aminobutyl)piperazine (FDKP) as aninhalation powder, a pharmacodynamic response, including endogenousinsulin production, reduction in glucagon and glucose levels, indiabetic animals can be elicited. In addition, the late surge in nativeGLP-1 linked to increased insulin secretion can be mimicked bypostprandial administration of GLP-1/FDKP inhalation powder.

A Phase 1a clinical trials of GLP-1/FDKP inhalation powder was designedto test the safety and tolerability of selected doses of a new inhaledglycemic control therapeutic product for the first time in humansubjects. GLP-1/FDKP inhalation powder was administered using theMedTone® Inhaler device, previously tested. The experiments weredesigned to identify the safety and tolerability of various doses ofGLP-1/FDKP inhalation powder by pulmonary inhalation. Doses wereselected for human use based on animal safety study results fromnon-clinical studies in rats and primates using GLP-1/FDKP administeredby inhalation as described in U.S. application Ser. No. 11/735,957,which is incorporated herein by reference.

Twenty-six subjects were enrolled into 5 cohorts to provide up to 4evaluable subjects in each of cohorts 1 and 2 and up to 6 evaluablesubjects in each of cohorts 3 to 5 who met eligibility criteria andcompleted the study. Each subject was dosed once with GLP-1 asGLP-1/FDKP inhalation powder at the following dose levels: cohort 1:0.05 mg; cohort 2: 0.45 mg; cohort 3: 0.75 mg; cohort 4: 1.05 mg andcohort 5: 1.5 mg of GLP-1. Dropouts were not replaced. These dosagesassumed a body mass of 70 kg. Persons of ordinary skill in the art candetermine additional dosage levels based on the studies disclosedherein.

In these experiments, the safety and tolerability of ascending doses ofGLP-1/FDKP inhalation powder in healthy adult male subjects weredetermined. The tolerability of ascending doses of GLP-1/FDKP inhalationpowder were determined by monitoring pharmacological or adverse effectson variables including reported adverse events (AE), vital signs,physical examinations, clinical laboratory tests and electrocardiograms(ECG).

Additional pulmonary safety and pharmacokinetic parameters were alsoevaluated. Pulmonary safety as expressed by the incidence of pulmonaryand other adverse events and changes in pulmonary function between Visit1 (Screening) and Visit 3 (Follow-up) was studied. Pharmacokinetic (PK)parameters of plasma GLP-1 and serum fumaryl diketopiperazine (FDKP)following dosing with GLP-1/FDKP inhalation powder were measured asAUC_(0-120 min) plasma GLP-1 and AUC_(0-480 min) serum FDKP. AdditionalPK parameters of plasma GLP-1 included the time to reach maximal plasmaGLP-1 concentration, T_(max) plasma GLP-1; the maximal concentration ofGLP-1 in plasma, C_(max) plasma GLP-1, and the half of total time toreach maximal concentration of GLP-1 in plasma, T_(1/2) plasma GLP-1.Additional PK parameters of serum FDKP included T_(max) serum FDKP,C_(max) serum FDKP, and T_(1/2) serum FDKP. Clinical trial endpointswere based on a comparison of the following pharmacological and safetyparameters determined in the trial subject population. Primary endpointsincluded the incidence and severity of reported AEs, including cough anddyspnea, nausea and/or vomiting, as well as changes from screening invital signs, clinical laboratory tests and physical examinations.Secondary endpoints included pharmacokinetic disposition of plasma GLP-1and serum FDKP (AUC_(0-120 min) plasma GLP-1 and AUC_(0-480 min) serumFDKP), plasma GLP-1 (T_(max) plasma GLP-1, C_(max) plasma GLP-1 T_(1/2)plasma GLP-1); serum FDKP (T_(max) serum FDKP, C_(max) serum FDKP);pulmonary function tests (PFTs), and ECG.

The clinical trial consisted of 3 clinic visits: 1) One screening visit(Visit 1); 2) One treatment visit (Visit 2); and 3) One follow-up visit(Visit 3) 8-14 days after Visit 2. A single dose of GLP-1/FDKPinhalation powder was administered at Visit 2.

Five doses of GLP-1/FDKP inhalation powder (0.05, 0.45, 0.75, 1.05 and1.5 mg of GLP-1) were assessed. To accommodate all doses, formulatedGLP-1/FDKP was mixed with FDKP inhalation powder containing particleswithout active agent. Single-dose cartridges containing 10 mg dry powderconsisting of GLP-1/FDKP inhalation powder (15% weight to weightGLP-1/FDKP) as is or mixed with the appropriate amount of FDKPinhalation powder was used to obtain the desired dose of GLP-1 (0.05 mg,0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg). The first 2 lowest dose levelswere evaluated in 2 cohorts of 4 subjects each and the 3 higher doselevels were evaluated in 3 cohorts of 6 subjects each. Each subjectreceived only 1 dose at 1 of the 5 dose levels assessed. In addition toblood drawn for GLP-1 (active and total) and FDKP measurements, sampleswere drawn for glucagon, glucose, insulin, and C-peptide determination.The results from these experiments are described with reference to thefollowing figures and tables.

FIG. 1 depicts the active GLP-1 plasma concentration in cohort 5 afterpulmonary administration of 1.5 mg of GLP-1 dose. The data showed thatthe peak concentration occurred prior to the first sampling point at 3minutes, closely resembling intravenous (IV) bolus administration. GLP-1plasma concentrations in some subjects were greater than 500 pmol/L, theassay limit. Peak active GLP-1 plasma concentrations range from about150 pmol/L to about 500 pmol/L. Intravenous bolus administration ofGLP-1 as reported in the literature (Vilsboll et al. 2000) results inratios of total:active GLP-1 of 3.0-5.0 compared to a ratio of 1.5 incohort 5 of this study. At comparable active concentrations themetabolite peaks were 8-9 fold greater following intravenousadministration compared to pulmonary administration, suggesting thatpulmonary delivery results in rapid delivery and less degradation ofGLP-1.

TABLE 1 Treatment 0.05 mg 0.45 mg 0.75 mg 1.05 mg 1.5 mg Parameter^(a)(n = 4) (n = 4) (n = 6) (n = 6) (n = 6) GLP-1^(a) AUC₀₋₁₂₀ ND n = 1 n =6 n = 4 n = 4 (min*pmol/L) 355.33 880.12 1377.88 AULQ (195.656)(634.054) C_(max) (pmol/L) n = 4 n = 4 n = 6 n = 6 n = 6 2.828 24.63081.172 147.613 310.700 (2.4507) (8.7291) (63.3601) (122.7014 (54.2431)t_(max) (min) n = 4 n = 4 n = 6 n = 6 n = 6 3.00 3.00 3.00 3.00 3.00(3.00, 3.00) (3.00, 4.02) (3.00, 6.00) (3.00, 4.98) (3.00, 3.00) T_(1/2)(min) n = 1 n = 3 n = 6 n = 4 n = 6 6.1507 3.0018 5.5000 3.6489 3.9410(0.83511) (2.96928) (1.88281) (1.79028) FDKP AUC₀₋₁₂₀ n = 6 n = 6(min*pmol/L) 22169.2 25594.7 (4766.858) (5923.689) C_(max) (pmol/L) n =6 n = 6 184.21 210.36 (56.893) (53.832) t_(max) (min) n = 6 n = 6 4.506.00 (3.00, 25.02) (3.00, 19.98) T_(1/2) (min) n = 6 n = 6 126.71 123.82(11.578) (15.640) ^(a)All parameters are mean (SD) except tmax, which ismedian (range) AULQ - Two or more subjects in the dose group had plasmaconcentrations of the analyte that were AULQ; NA = The pharmacokineticprofile did not meet the specifications for this profile because of theshort sampling time (20 minutes); ND = Parameter could not be calculatedbecause of insufficient data is some subjects.

In healthy individuals, physiological post-prandial venous plasmaconcentrations of GLP-1 typically range from 10-20 pmol/L (Vilsboll etal. J. Clin. Endocr. & Metabolism. 88(6):2706-13, June 2003). Theselevels were achieved with some subjects in cohort 2, who received 0.45mg GLP-1. Higher doses of GLP-1 produced peak plasma GLP-1concentrations substantially higher than physiological peak venousconcentrations. However, because the half-life of GLP-1 is short (about1-2 min), plasma concentrations of active GLP-1 fell to thephysiological range by 9 min after administration. Although the peakconcentrations are much higher than those seen physiologically in thevenous circulation, there is evidence that local concentrations of GLP-1may be much higher than those seen systemically.

Table 1 shows the pharmacokinetic profile of GLP-1 in a formulationcomprising FDKP from this study.

FDKP pharmacokinetic parameters are also represented in Table 1 forcohorts 4 and 5. Other cohorts were not analyzed. The data also showsthat mean plasma concentration of FDKP for the 1.05 mg and the 1.5 mgGLP-1 treated subjects were about 184 and 211 pmol/L, respectively.Maximal plasma FDKP concentrations were attained at about 4.5 and 6 minafter administration for the respective dose with a half-life about 2 hr(127 and 123 min).

FIG. 2A depicts mean insulin concentrations in subjects treated with aninhalable dry powder formulation of GLP-1 at a dose of 1.5 mg. The datashow the 1.5 mg GLP-1 dose induced endogenous insulin release fromβ-cells since insulin concentrations were detected in all subjects, andthe mean peak insulin concentrations of about 380 pmol/L occurred at 6min after dosing or earlier. The insulin release was rapid, but notsustained, since plasma insulin concentration fell rapidly after theinitial response to GLP-1. FIG. 2B depicts the GLP-1 plasmaconcentration of subjects treated with the 1.5 mg dose of GLPadministered by pulmonary inhalation compared to subcutaneousadministration of a GLP-1 dose. The data illustrates that pulmonaryadministration of GLP-1 occurs relatively fast and peak plasmaconcentration of GLP-1 occur faster than with subcutaneousadministration. Additionally, pulmonary inhalation of GLP-1 leads toGLP-1 plasma concentrations returning to basal levels much faster thanwith subcutaneous administration. Thus the exposure of the patient toGLP-1 provided by pulmonary inhalation using the present drug deliverysystem is shorter in time than by subcutaneous administration and thetotal exposure to GLP-1 as measured by AUC is less for the inhaledinsulin. FIG. 2C illustrates that pulmonary administration of a drypowder formulation of GLP-1 induces an insulin response which is similarto the response obtained after intravenous administration of GLP-1, butdifferent in peak time and amount of endogenous insulin produced thanwith subcutaneous GLP-1 administration, which indicates that pulmonaryadministration of GLP-1 using the present formulation is moreefficacious at inducing an insulin response.

FIG. 3 depicts the plasma C-peptide concentrations in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation. The data demonstrate thatC-peptide is released following GLP-1 inhalation confirming endogenousinsulin release.

FIG. 4 depicts fasting plasma glucose concentrations in subjects treatedwith the GLP-1 formulation containing GLP-1. Mean fasting plasma glucose(FPG) concentrations were approximately 4.7 mmol/L for the 1.5 mg GLP-1treated subjects. GLP-1 mediated insulin release is glucose dependent.Hypoglycemia is not historically observed in euglycemic subjects. Inthis experiment, the data clearly show that glucose concentrations inthese euglycemic healthy subjects were reduced following pulmonaryadministration of GLP-1. At the 1.5 mg GLP-1 dose, two of the sixsubjects had glucose concentrations lowered by GLP-1 to below 3.5mmol/L, the laboratory value that defines hypoglycemia. Plasma glucosedecreased more than 1.5 mol/L in two of the six subjects that receivedthe 1.5 mg GLP-1 dose. Moreover, decreases in plasma glucose werecorrelated to the GLP-1 dose. The smallest decrease in glucoseconcentration was seen with the 0.05 mg dose, and the largest decreasewas seen with the 1.5 mg dose. The three intermediate doses of GLP-1produced roughly equal decreases in plasma glucose. The data indicatethat the GLP-1 glucose-dependency was overcome based on GLP-1concentrations above the physiologic range. Physiologic ranges for GLP-1(7-36) amide in normal individuals has been reported to be in the rangeof 5-10 pmol/L during fasting, and increase rapidly after eating to 15to 50 pmol/L (Drucker, D. and Nauck, M. The Lancet 368:1696-1705, 2006).

FIG. 5 further depicts insulin concentrations in plasma after GLP-1pulmonary administration are dose dependent. In most subjects, theinsulin release was not sustained, since plasma insulin concentrationfell rapidly after the initial response to GLP-1 administration. In mostsubjects, the peak plasma insulin response ranged from 200-400 pmol/Lwith one subject exhibiting peak plasma insulin levels that exceeded 700pmol/L. Thus, the data indicate that insulin response is GLP-1 dosedependent.

FIG. 6 depicts glucagon concentrations in plasma after GLP-1 pulmonaryadministration at the various dosing groups. Baseline glucagon levelsranged from 13.2 pmol/L to 18.2 pmol/L in the various dose groups. Themaximum change in plasma glucagon was seen at 12 min after dosing. Thelargest decrease in plasma glucagon was approximately 2.5 pmol/L and wasseen in the 1.5 mg dose group. The maximum suppression of glucagonsecretion was potentially underestimated because the minima did notalways occur at 12 min.

Tables 2 and 3 report the adverse events or side effect symptomsrecorded for the patient population in the study. The list of adverseevents reported in the literature for GLP-1 administered by injection isnot extensive; and those reported have been described as mild ormoderate, and tolerable. The primary adverse events reported have beenprofuse sweating, nausea and vomiting when active GLP-1 concentrationsexceed 100 pmol/L. As shown in Tables 1 and 3, and FIG. 1, pulmonaryadministration at doses of 1.05 mg and 1.5 mg resulted in active GLP-1concentrations greatly exceeding 100 pmol/L without the side effectsnormally observed with parenteral (subcutaneous, intravenous [eitherbolus or infusion]) GLP-1. None of the subjects in this study reportedsymptoms of nausea, profuse sweating or vomiting. Subjects in Cohort 5reached C_(max) comparable to that observed with a 50 μg/kg IV bolusdata (reported by Vilsboll et al. 2000), where the majority of subjectsreported significant adverse events.

TABLE 2 Adverse Events 0.05 mg 0.45 mg 0.75 mg 1.05 mg 1.5 mg AdverseEvent (n = 4) (n = 4) (n = 6) (n = 6) (n = 6) Cough 3 1 3 5 5 Dysphonia2 — 2 3 3 Productive Cough — — 1 — — Throat Irritation — — — 1 —Headache 1 1 — 1 1 Dizziness — — — — 2 Dysgeusia — — 1 — — Fatigue — — 11 1 Seasonal Allergy — — — 1 — Rhinitis — — — 1 — Increased Appetite — —— — 1

TABLE 3 Comparative Adverse Events of GLP-1: IV vs. PulmonaryAdministration IV^(†) IV^(†)* Pulmonary* Adverse Events (16.7 μg) (50μg) (1.5 mg) Reduced well-being 42% 100%  17%  Nausea 33% 83% 0% Profusesweating 17% 67% 0% ^(†)Vilsboll et al. Diabetes Care, June 2000;*Comparable C_(max)

Tables 2 and 3 show there were no serious or severe adverse eventsreported by any subjects in the study who received GLP-1 by pulmonaryinhalation. The most commonly reported adverse events were thoseassociated with inhalation of a dry powder, cough and throat irritation.Surprisingly, in the patients treated by pulmonary inhalation, nosubject reported nausea or dysphoria, and there was no vomitingassociated with any of these subjects. The inventors also found thatpulmonary administration of GLP-1 in a dry powder formulation lackinhibition of gastric emptying in the above subjects (data not shown).Inhibition of gastric emptying is a commonly encountered unwanted sideeffect associated with injected standard formulations of GLP-1.

In summary, the clinical GLP-1/FDKP powder contained up to 15 wt % GLP-1providing a maximum dose of 1.5 mg GLP-1 in 10 mg of powder. Andersencascade measurements indicated that 35-70% of the particles hadaerodynamic diameters <5.8 μm. A dose of 1.5 mg GLP-1 produced mean peakconcentrations >300 pmol/L of active GLP-1 at the first sampling time (3min); resulted in mean peak insulin concentrations of 375 pmol/L at thefirst measured time point (6 min); reduced mean fasting plasma glucosefrom 85 to 70 mg/dL 20 min after dosing; and was well tolerated and didnot cause nausea or vomiting.

Example 2 Comparison of Pulmonary Administration of GLP-1 and Exenatide,and Subcutaneous Administration of Exenatide to Male Zucker DiabeticFatty Rats

Much effort has been expended in developing analogs of GLP-1 with longercirculating half-lives to arrive at a clinically useful treatment. Asdemonstrated herein pulmonary administration of GLP-1 also providesclinically meaningful activity. It was thus of interest to compare thesetwo approaches.

Preparation of FDKP Particles.

Fumaryl diketopiperazine (FDKP) and polysorbate 80 were dissolved indilute aqueous ammonia to obtain a solution containing 2.5 wt % FDKP and0.05 wt % polysorbate 80. The FDKP solution was then mixed with anacetic acid solution containing polysorbate 80 to form particles. Theparticles were washed and concentrated by tangential flow filtration toachieve approximately 11% solids by weight.

Preparation of GLP-1 Stock Solution.

A 10 wt % GLP-1 stock solution was prepared in deionized water bycombining 60 mg GLP-1 solids (86.6% peptide) with 451 mg deionizedwater. About 8 μL glacial acetic acid was added to dissolve the peptide.

Preparation of GLP-1/FDKP Particles.

A 1 g portion of the stock FDKP suspension (108 mg particles) wastransferred to a 2 mL polypropylene tube. The appropriate amount ofGLP-1 stock solution (Table 1) was added to the suspension and gentlymixed. The pH of the suspension was adjusted from pH ˜3.5 to pH ˜4.5 byadding 1 μL aliquot of 50% (v/v) ammonium hydroxide. The GLP-1/FDKPparticle suspension was then pelleted into liquid nitrogen andlyophilized. The dry powders were analyzed by high performance liquidchromatography (HPLC) and found comparable to theoretical values.

Preparation of Exenatide Stock Solution.

A 10 wt % exendin stock solution was prepared in 2% wt acetic acid bycombining 281 mg exendin solids (88.9% peptide) with 2219 mg 2% wtacetic acid.

Preparation of Exenatide/FDKP Particles.

A 1533 mg portion of a stock FDKP particle suspension (171 mg particles)was transferred to a 4 mL glass vial. A 304 mg portion of exendin stocksolution was added to the suspension and gently mixed. The pH of thesuspension was adjusted from pH ˜3.7 to pH ˜4.5 by adding 3-5 μLaliquots of 25% (v/v) ammonium hydroxide. The exenatide/FDKP particlesuspension was then pelleted into liquid nitrogen and lyophilized. Thedry powders were analyzed by high performance liquid chromatography(HPLC) and found comparable to theoretical values.

Pharmacokinetic and Pharmacodynamic Assessment in Rats.

Male Zucker Diabetic Fatty (ZDF) rats (5/group) were assigned to one offour test groups. Animals were fasted overnight then administeredglucose (1 g/kg) by intraperitoneal injection immediately prior to testarticle dosing. Animals in the control group received air by pulmonaryinsufflation. Animals in Group 1 received exenatide (0.3 mg) in saline(0.1 mL) by subcutaneous (SC) injections. Animals in Group 2 received15% by weight exenatide/FDKP (2 mg) by pulmonary insufflation. Animalsin Group 3 received 15% by weight GLP-1/FDKP (2 mg) by pulmonaryinsufflation. Blood samples were collected from the tail prior to dosingand at 15, 30, 45, 60, 90, 120, 240, and 480 min after dosing. Plasmawas harvested. Blood glucose and plasma GLP-1 or plasma exenatideconcentrations were determined.

Exenetide pharmacokinetics are reported in FIG. 7A. These data showedthat exenetide is absorbed rapidly following insufflation ofexenetide/FDKP powder. The bioavailability of the inhaled exenetide was94% compared to subcutaneous injection. This may indicate that pulmonaryadministration is particularly advantageous to exenatide. The time tomaximum peak circulating exenetide concentrations (T_(max)) was 30 minin rats receiving subcutaneous exenetide compared to <15 min in ratsreceiving inhaled exenetide. This T_(max) was similar to that ofinsufflated GLP-1/FDKP (data not shown).

Comparative pharmacodynamics are reported in FIG. 8. These data showedthe changes in blood glucose for all four test groups. Glucoseexcursions following the glucose tolerance test were lower in animalsreceiving inhaled exenetide/FDKP as compared to animals receiving SCexenetide. Since exenetide exposure was comparable in both groups (FIG.7), these data suggest that the shorter time to peak exenetideconcentrations in the exenetide/FDKP group provided better glucosecontrol. Additionally, glucose excursions were comparable in animalsreceiving either GLP-1/FDKP or exenetide/FDKP. These data are surprisingbecause the circulating half-life of exenetide (89 min) is considerablylonger than that of GLP-1 (15 min). Indeed, exenetide was developed tomaximize circulating half-life for the purpose of increasing efficacy.These data suggest that the longer circulating half-life of exenetideoffers no advantage in controlling hyperglycemia when using pulmonaryadministration. Moreover pulmonary administration of either moleculeprovided superior blood glucose control the SC exenatide.

FIG. 7 depicts mean plasma exendin concentrations in male ZDF ratsreceiving exendin-4/FDKP powder formulation administered by pulmonaryinsufflation versus subcutaneous exendin-4. The closed squares representthe response following pulmonary insufflation of exendin-4/FDKP powder.The open squares represent the response following administration ofsubcutaneously administered exendin-4. The data are plotted as ±standarddeviation. The data show that rats insufflated with powders providingGLP-1 doses of 0.12, 0.17, and 0.36 mg produced maximum plasma GLP-1concentrations (C_(max)) of 2.3, 4.9 and 10.2 nM and exposures (AUC) of57.1 nM·min, 92.6 nM·min, and 227.9 nM·min, respectively (t_(max)=10min, t_(1/2)=10 min). In an intraperitoneal glucose tolerance testconducted after 4 consecutive days of dosing 0.3 mg GLP-1 per day,treated animals exhibited significantly lower glucose concentrationsthan the control group (p<0.05). At 30 min post-challenge, glucoseincreased by 47% in control animals but only 17% in treated animals.

FIG. 8 depicts the change in blood glucose from baseline in male ZDFrats receiving either air control, exendin-4/FDKP powder, or GLP-1/FDKPpowder via pulmonary insufflation versus subcutaneous exendin-4 andexendin-4 administered by pulmonary insufflation. The closed diamondsrepresent the response following pulmonary insufflation ofexendin-4/FDKP powder. The closed circles represent the responsefollowing administration of subcutaneous exendin-4. The closed trianglesrepresent the response following administration of GLP-1/FDKP powder.The closed squares represent the response following pulmonaryinsufflation of air alone. The open squares represent the response givenby 2 mg of GLP-1/FDKP given to the rats by insufflation followed by a 2mg exendin-4/FDKP powder administered also by insufflation.

Example 3 Oxyntomodulin/FDKP Powder Preparation

Oxyntomodulin, also known as glucagon-37, is a peptide consisting of 37amino acid residues. The peptide was manufactured and acquired fromAmerican Peptide Company, Inc. of Sunnyvale, Calif. FDKP particles insuspension were mixed with an oxyntomodulin solution, then flash frozenas pellets in liquid nitrogen and lyophilized to produce sample powders.

Six powders were prepared with target peptide content between 5% and30%. Actual peptide content determined by HPLC was between 4.4% and28.5%. The aerodynamic properties of the 10% peptide-containing powderwere analyzed using cascade impaction.

The FDKP solution was then mixed with an acetic acid solution containingpolysorbate 80 to form particles. The particles were washed andconcentrated by tangential flow filtration to achieve approximately 11%solids by weight.

FDKP particle suspension (1885 mg×11.14% solids=210 mg FDKP particles)was weighed into a 4 mL clear glass vial. The vial was capped and mixedusing a magnetic stirrer to prevent settling. Oxyntomodulin solution(909 mg of 10% peptide in 2 wt % acetic acid) was added to the vial andallowed to mix. The final composition ratio was approximately 30:70oxyntomodulin:FDKP particles. The oxyntomodulin/FDKP suspension had aninitial pH of 4.00 which was adjusted to pH 4.48 by adding 2-10 μLincrements of 1:4 (v/v) ammonium hydroxide/water. The suspension waspelleted into a small crystallization dish containing liquid nitrogen.The dish was placed in a freeze dryer and lyophilized at 200 mTorr. Theshelf temperature was ramped from −45° C. to 25° C. at 0.2° C./min andthen held at 25° C. for approximately 10 hours. The resultant powder wastransferred to a 4 mL clear glass vial. Total yield of the powder aftertransfer to the vial was 309 mg (103%). Samples were tested foroxyntomodulin content by diluting the oxyntomodulin preparation insodium bicarbonate and assaying by high pressure liquid chromatographyin a Waters 2695 separations system using deionized with 0.1%trifluoroacetic acid (TFA) and acetonitrile with 0.1% TFA as mobilephases, with the wavelength detection set at 220 and 280 nm. Data wasanalyzed using a Waters Empower™ software program.

Pharmacokinetic and Pharmacodynamic Assessment in Rats.

Male ZDF rats (10/group) were assigned to one of four groups. Animals inthe one group received oxyntomodulin by intravenous injection. Animalsin the other three groups received 5% oxyntomodulin/FDKP powder(containing 0.15 mg oxyntomodulin), 15% oxyntomodulin/FDKP powder(containing 0.45 mg oxyntomodulin), or 30% oxyntomodulin/FDKP powder(containing 0.9 mg oxyntomodulin) by pulmonary insufflation. Bloodsamples were collected from the tail prior to dosing and at varioustimes after dosing for measurement of plasma oxyntomodulinconcentrations (FIG. 9A). Food consumption was also monitored at varioustimes after dosing with oxyntomodulin (FIG. 9B).

FIG. 9A is a graph comparing the plasma concentrations of oxyntomodulinfollowing administration of an inhalable dry powder formulation atvarious amounts in male ZDF rats and control rats receivingoxyntomodulin by intravenous injection. These data show thatoxyntomodulin is absorbed rapidly following insufflation ofoxyntomodulin/FDKP powder. The time to maximum peak circulatingoxyntomodulin concentrations (T_(max)) was less than 15 min in ratsreceiving inhaled oxyntomodulin. This study shows that the half life ofoxyntomodulin is from about 22 to about 25 min after pulmonaryadministration.

FIG. 9B is a bar graph showing cumulative food consumption in male ZDFrats treated with intravenous oxyntomodulin or oxyntomodulin/FDKP powderadministered by pulmonary insufflation compared to control animalsreceiving an air stream. The data show that pulmonary administration ofoxyntomodulin/FDKP reduced food consumption to a greater extent thaneither intravenous oxyntomodulin or air control with a single dose.

In a similar set of experiments, rats received an air stream as control(Group 1) or 30% oxyntomodulin/FDKP powder by pulmonary insufflation.Rats administered oxyntomodulin/FDKP inhalation powder received doses ofeither 0.15 mg oxyntomodulin (as 0.5 mg of oxyntomodulin/FDKP powder;Group 2), 0.45 mg oxyntomodulin (as 1.5 mg of oxyntomodulin/FDKP powder,Group 3) or 0.9 mg oxyntomodulin (as 3 mg of oxyntomodulin/FDKP powder,Group 4) prepared as described above. The studies were conducted in ZDFrats fasted for 24 hr prior to the start of the experiment. Rats wereallowed to eat after receiving the experimental dose. A predeterminedamount of food was given to the rats and the amount of food the ratsconsumed was measured at various times after the start of theexperiment. The oxyntomodulin/FDKP dry powder formulation wasadministered to the rats by pulmonary insufflation and food measurementsand blood samples were taken at various points after dosing.

FIGS. 10A and 10B show circulating oxyntomodulin concentrations for alltest animals and the change in food consumption from control,respectively. Rats given oxyntomodulin consumed significantly less foodthan the control rats for up to 6 hr after dosing. Higher doses ofoxyntomodulin appeared to suppress appetite more significantly that thelower doses indicating that appetite suppression is dose dependent, asthe rats given the higher dose consumed the least amount of food at alltime points measured after dosing.

Maximal concentrations of oxyntomodulin in blood were detected at 10 to30 min and that maximal concentration of oxyntomodulin was dosedependent as the rats receiving 1.5 mg of oxyntomodulin had a maximalplasma concentration of 311 μg/mL and rats receiving 3 mg ofoxyntomodulin had a maximal plasma concentration of 660 μg/mL. Thehalf-life (t_(1/2)) of oxyntomodulin in the Sprague Dawley rats afteradministration by pulmonary insufflation ranged from about 25 to 51 min.

Example 4 Administration of GLP-1 in an Inhalable Dry Powder to Type 2Diabetic Patients

A Phase 1 clinical trial of GLP-1/FDKP inhalation powder was conductedin patients suffering with Type 2 diabetes mellitus to assess theglucose levels of the patients before and after treatment with GLP-1 drypowder formulation by pulmonary inhalation. These studies were conductedaccording to Example 1 and as described herein. GLP-1 inhalation powderwas prepared as described in U.S. patent application Ser. No.11/735,957, which disclosure is incorporated herein by reference. Thedry inhalation powder contained 1.5 mg of human GLP-1 (7-36) amide in atotal of 10 mg dry powder formulation containing FDKP in single dosecartridge. For this study, 20 patients with Type 2 diabetes, includingadult males and postmenopausal females, were fasted overnight andremained fasted for a period of 4 hr after GLP-1 inhalation powderadministration. The dry powder formulation was administered using theMedtone® dry powder inhaler (MannKind Corporation), and described inU.S. patent application Ser. No. 10/655,153, which disclosure isincorporated herein by reference in its entirety.

Blood samples for assessing serum glucose levels from the treatedpatients were obtained at 30 min prior to dosing, at dosing (time 0),and at approximately 2, 4, 9, 15, 30, 45, 60, 90, 120 and 240 minfollowing GLP-1 administration. The serum glucose levels were analyzedfor each sample.

FIG. 11 is a graph showing the results of these studies and depicts theglucose values obtained from six fasted patients with Type 2 diabetesfollowing administration of a single dose of an inhalable dry powderformulation containing GLP-1 at various time points. The glucose valuesfor all six patients decreased following administration of GLP-1 andremained depressed for at least 4 hrs after administration at thetermination of the study.

FIG. 12 is a graph showing the mean glucose values for the group of sixfasted patients with Type 2 diabetes whose glucose values are shown inFIG. 11. In FIG. 12, the glucose values are expressed as the mean changeof glucose levels from zero time (dosing) for all six patients. FIG. 12shows a mean glucose drop of approximately 1 mmol/L, which isapproximately equivalent to from about 18 mg/dL to about 20 mg/dL, isattained by the 30 min time point. This mean drop in glucose levels tolast for 120 min. The changes are larger in subjects with higherbaseline glucose and more prolonged, whereas in 2 of the 6 subjects,those subjects with the lowest baseline fasted blood glucose, showedonly a transient lowering of glucose levels in this timeframe (data notshown). It was noted that those with higher fasting glucose do nottypically have the same insulin response as those with lower values, sothat when stimulated, those subjects with higher fasting glucosetypically exhibit a greater response than those whose glucose value arecloser to normal.

Example 5 First Pass Distribution Model of Intact GLP-1 to Brain andLiver

First pass distribution of GLP-1 through the systemic circulationfollowing pulmonary delivery and intravenous bolus administration wascalculated to determine the efficacy of delivery for both methods ofGLP-1 administration. A model was developed based on the assumptionsthat: (1) the absorption of GLP-1 from the lungs and into the pulmonaryveins exhibited zero-order kinetics; (2) the distribution of GLP-1 tothe brain and within the brain occurs instantaneously, and (3) clearanceof GLP-1 from the brain and liver distribution is driven by basal bloodflow only. Based on these assumptions, the analysis to determine theamount of GLP-1 in the brain and liver was based on published data withrespect to extraction of GLP-1 by certain tissues and organs (Deacon, C.F. et al. “Glucagon-like peptide 1 undergoes differentialtissue-specific metabolism in the anesthetized pig.” AmericanPhysiological Society, 1996, pages E458-E464), and blood flowdistribution to the body and rate due to cardiac output from humanstudies (Guyton Textbook of Physiology, 10^(th) Edition; W. B. Saunders,2000, page 176). In a normal subject (70 kg) having normal physiologicalparameters such as blood pressure at resting, the basal flow rate to thebrain and liver are 700 mL/min and 1350 mL/min, respectively. Based oncardiac output, blood flow distribution to the body has been calculatedto be 14% to the brain, 27% to the liver and 59% to remaining bodytissues (Guyton).

Using the above-mentioned parameters, the relative amounts of GLP-1 thatwould be distributed to the brain and liver for a 1 mg dose given bypulmonary and intravenous administration were determined. One mg ofGLP-1 was divided by 60 seconds, and the resultant number was multipliedby 14% flow distribution to the brain. Therefore, every second afraction of the dose is appearing in the brain. From the data availableindicating that blood in the brain is equal to 150 mL and the clearancerate is 700 mL/min, the calculations on clearance of GLP-1 yields about12 mL/second, which equals approximately 8% of the blood volume beingcleared from the brain per second. In the intravenous studies in pigsreported by Deacon et al., 40% of GLP-1 was instantaneously metabolizedin the vein and 10% was also metabolized in the deoxygenated blood inthe lung. Accordingly, 40% followed by another 10% of the total GLP-1was subtracted from the total amount administered in the calculationswith respect to the intravenous data analysis.

For the GLP-1 amounts estimated in the liver, the same degradationassumptions were made for the intravenous and pulmonary administrationroutes, with 40% followed by another 10% total amount loss for the IVdose. Twenty-seven percent of the remaining GLP-1 was assumed to bedistributed to the liver, with 75% of the blood passing through theportal bed first. Instantaneous distribution of blood in the liver wasassumed. Calculations were as follows: One mg of GLP-1 was divided by 60seconds, 40% followed by another 10% of the total GLP-1 was subtractedfrom the total amount administered with respect to the intravenous dataanalysis. No degradation was assumed for the pulmonary administration.The resultant numbers were multiplied by 27% flow distribution to theliver, for both routes of administration, with 75% of this amountpassing though the portal bed first. In the intravenous studies in pigsreported by Deacon et al., 20% extraction by the portal bed wasreported; hence 75% of the amount of GLP-1 was reduced by 20% beforebeing introduced into the liver. Therefore, the total amount of GLP-1appearing in the liver every second is comprised of a fraction which hasundergone metabolism in the portal bed. From the data availableindicating that blood volume in the liver is equal to 750 mL and theclearance rate is 1350 mL/minute, the calculations on clearance of GLP-1yields about 22.5 mL/second, which equals approximately 3% of the bloodvolume being cleared from the liver per second. Deacon et al. reported45% degradation in the liver, accordingly, 45% of the total GLP-1 wassubtracted from the total amount appearing in the liver, and theremainder was added to the total remaining amount.

The results of the calculations described above are presented in Tables4 and 5. The calculated GLP-1 distribution in brain and liver afterpulmonary administration (Table 4) are shown below:

TABLE 4 Pulmonary administration of 1 mg GLP-1 Time in Seconds Brain(μg) Liver (μg) 1 2.3 2.10 5 9.94 9.91 60 29.0 58.9

The results illustrating the distribution of GLP-1 after an intravenousbolus administration are shown in Table 5 below:

TABLE 5 Intravenous bolus administration of 1 mg GLP-1 over 1 minuteTime in Seconds Brain (μg) Liver (μg) 1 1.26 1.14 5 5.37 5.35 60 15.631.7

The data above are representative illustrations of the distribution ofGLP-1 to specific tissues of the body after degradation of GLP-1 byendogenous enzymes. Based on the above determinations, the amounts ofGLP-1 in brain and liver after pulmonary administration are about 1.82to about 1.86 times higher than the amounts of after intravenous bolusadministration. Therefore, the data indicate that pulmonary delivery ofGLP-1 can be a more effective route of delivery when compared tointravenous administration of GLP-1, as the amount of GLP-1 at varioustimes after administration would be about double the amount obtainedwith intravenous administration. Therefore, treatment of a disease ordisorder comprising GLP-1 by pulmonary administration would requiresmaller total amounts, or almost half of an intravenous GLP-1 dose thatis required to yield the same or similar effects.

Example 6

The studies in this example were conducted to measure thepharmacokinetic parameters of various active agents by subcutaneousadministration and in formulations comprising a FDKP, FDKP disodiumsalt, succinyl-substituted-DKP (SDKP, also referred to herein asCompound 1) or asymmetrical (fumaryl-monosubstituted)-DKP (also referredherein as Compound 2) to ZDF rats administered by pulmonaryinsufflation. The rats were divided into 8 groups and five rats wereassigned to each group. Each rat in Group 1 received a 0.3 mg dose ofexendin-4 in phosphate buffered saline solution by pulmonary liquidinstillation; Group 2 received 0.3 mg of exendin-4 in phosphate bufferedsaline by subcutaneous injection.

Rats in Groups 3-8 received their dosing of active agent or exendin-4 bypulmonary insufflation as follows: Group 3 rats received a 2 mgformulation of GLP-1/FDKP by pulmonary insufflation, followed by a 2 mgdose of exendin-4; Group 4 received a formulation of exendin-4/FDKP;Group 5 rats received a 3 mg dose of exendin-4 formulated as a 9.2% loadin a disodium salt of FDKP; Group 6 rats received a 2 mg dose ofexendin-4 formulated as a 13.4% load in a disodium salt of FDKP; Group 7rats received a 2 mg dose of exendin-4 formulated as a 14.5% load inSDKP, and Group 8 rats received a 2 mg dose of exendin-4 formulated as a13.1% load in asymmetrical (fumaryl-mono-substituted) DKP.

The dosing of the animals occurred over the course of two days toaccommodate the high numbers of subjects. The various test articles wereadministered to the animals and blood samples were taken at varioustimes after dosing. Exendin-4 concentrations were measured in plasmaisolates; the results for which are provided in FIG. 13. As depicted inthe graph, Group 4 treated rats which received exendin-4 in aformulation containing FDKP exhibited high levels of exendin-4 in theblood earlier than 30 min and at higher levels than the rats in Group 2,which received exendin-4 by subcutaneous administration. In all groups,the levels of exendin-4 decrease sharply at about an hour afteradministration.

Administration of exendin-4/FDKP by pulmonary insufflation in ZDF ratshas similar dose-normalized C_(max), AUC, and bioavailability asexendin-4 administered as a subcutaneous injection. Exendin-4/FDKPadministered by pulmonary insufflation showed a greater than two-foldhalf life compared to exendin-4 by subcutaneous injection. Exendin-4administered as an fumaryl(mono-substituted)DKP, or SDKP formulationshowed lower dose normalized C_(max), AUC, and bioavailability comparedto subcutaneous injection (approximately 50% less) but higher levelsthan pulmonary instillation.

After an overnight fast, ZDF rats were given a glucose challenge byintraperitoneal injection (IPGTT). Treatment with exendin-4/FDKP showeda greater reduction in blood glucose levels following the IPGTT comparedto exendin-4 by the subcutaneous route. Compared to air control animals,blood glucose levels were significantly lowered following an IPGTT for30 and 60 min in animals administered exendin-4 by subcutaneousinjection and exendin-4/FDKP powder by pulmonary administration,respectively. Group 3 ZDF rats treated with exendin-4/FDKP and GLP-1 bypulmonary insufflation after treatment with intraperitoneal glucoseadministration (IPGTT) showed surprisingly lower blood glucose levelsfollowing IPGTT compared to either treatment alone at 30 min post dose(−28% versus −24%).

Example 7

The studies in this example were conducted to measure thepharmacokinetic and pharmacodynamic profile of peptide YY(3-36)formulations by pulmonary administration to ZDF rats compared tointravenous injections.

Preparation of PYY/FDKP formulation for pulmonary delivery: PeptideYY(3-36) (PYY) used in these experiments was obtained from AmericanPeptide and was adsorbed onto FDKP particles as a function of pH. A 10%peptide stock solution was prepared by weighing 85.15 mg of PYY into an8 ml clear vial and adding 2% aqueous acetic acid to a final weight of762 mg. The peptide was gently mixed to obtain a clear solution. FDKPsuspension (4968 mg, containing 424 mg of FDKP preformed particles) wasadded to the vial containing the PYY solution, which formed a PYY/FDKPparticle suspension. The sample was placed on a magnetic stir-plate andmixed thoroughly throughout the experiment. A micro pH electrode wasused to monitor the pH of the mixture. Aliquots of 2-3 μL of a 14-15%aqueous ammonia solution were used to incrementally increase the pH ofthe sample. Sample volumes (75 μL for analysis of the supernatant; 10 μLfor suspension) were removed at each pH point. The samples forsupernatant analysis were transferred to 1.5 ml, 0.22 μm filter tubesand centrifuged. The suspension and filtered supernatant samples weretransferred into HPLC autosampler vials containing 990 μL of 50 mMsodium bicarbonate solution. The diluted samples were analyzed by HPLCto assess the characteristics of the preparations. The experimentsindicated that, for example, a 10.2% of PYY solution can be adsorbedonto FDKP particles at pH 4.5 In this particular preparation, forexample, the PYY content of the resultant powder was determined by HPLCto be 14.5% (w/w). Cascade measurements of aerodynamic characteristicsof the powder showed a respirable fraction of 52% with a 98% cartridgeemptying when discharged through the MedTone® dry powder inhaler(MannKind Corporation). Based on the results above, multiple samplepreparations of PYY/FDKP powder were made, including, 5%, 10%, 15% and20% PYY.

Pharmacokinetic and pharmacodynamic studies: Female ZDF rats were usedin these experiments and divided into 7 groups; five rats were assignedto each group, except for Group 1 which had 3 rats. The rats were fastedfor 24 hr prior to being given their assigned dose and immediatelyprovided with food after dosing and allowed to eat as desired for theperiod of the experiment. Each rat in Group 1 received a 0.6 mg IV doseof PYY in phosphate buffered saline solution; Group 2 rats received 1.0mg of PYY pulmonary liquid instillation; Group 3 rats were designated ascontrol and received a stream of air; Groups 4-7 rats received a drypowder formulation for inhalation administered by pulmonary insufflationas follows: Group 4 rats received 0.15 mg of PYY in a 3 mg PYY/FDKPpowder formulation of 5% PYY (w/w) load; Group 5 rats received 0.3 mg ofPYY in a 3 mg PYY/FDKP powder formulation of 10% PYY (w/w) load; Group 6rats received 0.45 mg of PYY in a 3 mg PYY/FDKP powder formulation of15% PYY (w/w) load; Group 7 rats received 0.6 mg of PYY in a 3 mgPYY/FDKP powder formulation of 20% PYY (w/w) load.

Food consumption was measured for each rat at 30, 60, 90, 120, 240 minand 24 hr after dosing. PYY plasma concentrations and glucoseconcentrations were determined for each rat from blood samples takenfrom the rats before dosing and at 5, 10, 20, 30, 45, 60 and 90 minafter dosing. The results of these experiments are shown in FIGS. 14-16and Table 6 below. FIG. 14 is a bar graph of representative data fromexperiments measuring food consumption in female ZDF rats receiving PYYformulations by intravenous administration and by pulmonaryadministration in a formulation comprising a fumaryl-diketopiperazine atthe various doses. The data show that food consumption was reduced forall PYY-treated rats when compared to control with the exception ofGroup 2 which received PYY by instillation. Reduction in foodconsumption by the rats was statistically significant for the ratstreated by pulmonary insufflation at 30, 60, 90 and 120 min afterPYY-dosing when compared to control. The data in FIG. 14 also show thatwhile IV administration (Group 1) is relatively effective in reducingfood consumption in the rats, the same amount of PYY (0.6 mg)administered by the pulmonary route in an FDKP formulation (Group 7) wasmore effective in reducing the amount of food intake or suppressingappetite for a longer period of time. All PYY-treated rats receivingpulmonary PYY-FDKP powders consumed less food when compared to controls.

FIG. 15 depicts the measured blood glucose levels in the female ZDF ratsgiven PYY formulations by IV administration; by pulmonary administrationwith various formulations comprising a fumaryl-diketopiperazine and aircontrol rats. The data indicate the blood glucose levels of thePYY-treated rats by pulmonary insufflation remained relatively similarto the controls, except for the Group 1 rats which were treated with PYYIV. The Group 1 rats showed an initial lower blood glucose level whencompared to the other rats up to about 15 min after dosing.

FIG. 16 depicts representative data from experiments measuring theplasma concentration of PYY in the female ZDF rats given PYYformulations by IV administration; by pulmonary administration withvarious formulations comprising a fumaryl-diketopiperazine, and aircontrol rats taken at various times after administration. Thesemeasurements are also represented in Table 6. The data show that Group 1rats which were administered PYY IV attained a higher plasma PYYconcentration (30.7 μg/mL) than rats treated by pulmonary insufflation.Peak plasma concentration (T_(max)) for PYY was about 5 min for Groups1, 6 and 7 rats and 10 min for Group 2, 4 and 5 rats. The data show thatall rats treated by pulmonary insufflation with a PYY/FDKP formulationhad measurable amounts of PYY in their plasma samples, however, theGroup 7 rats had the highest plasma PYY concentration (4.9 μg/mL) andvalues remained higher than the other groups up to about 35 min afterdosing. The data also indicate that the plasma concentration of PYYadministered by pulmonary insufflation is dose dependent. Whileadministration by IV injection led to higher venous plasma concentrationof PYY that did pulmonary administration of PYY/FDKP at the dosagesused, the greater suppression of food consumption was nonethelessachieved with pulmonary administration of PYY/FDKP.

TABLE 6 Rat Group T½ Tmax Cmax AUCall/D Number (min) (min) (μg/mL)(min/mL) BA (%) 1 13 5 30.7 0.61  100% 2 22 10 1.7 0.06 11 4 23 10 0.510.10 16 5 30 10 1.33 0.15 25 6 26 5 2.79 0.20 33 7 22 5 4.90 0.22 36

FIG. 17 illustrates the effectiveness of the present drug deliverysystem as measured for several active agents, including insulin,exendin, oxyntomodulin and PYY and exemplified herewith. Specifically,FIG. 17 demonstrates the relationship between drug exposure andbioeffect of the pulmonary drug delivery system compared to IV and SCadministration of the aforementioned active agents. The data in FIG. 17indicate that the present pulmonary drug delivery system provides agreater bioeffect with lesser amounts of drug exposure than intravenousor subcutaneous administration. Therefore, lesser amounts of drugexposure can be required to obtain a similar or greater effect of adesired drug when compared to standard therapies. Thus, in oneembodiment, a method of delivering an active agent, including, peptidessuch as GLP-1, oxyntomodulin, PYY, for the treatment of disease,including diabetes, hyperglycemia and obesity which comprisesadministering to a subject in need of treatment an inhalable formulationcomprising one or more active agents and a diketopiperazine whereby atherapeutic effect is seen with lower exposure to the active agent thanrequired to achieve a similar effect with other modes of administration.In one embodiment, the active agents include peptides, proteins,lipokines.

Example 8 Assessment of GLP-1 Activity in Postprandial Type 2 DiabetesMellitus

The purpose of this study was to evaluate the effect of a GLP-1 drypowder formulation on postprandial glucose concentration and assess itssafety including adverse events, GPL-1 activity, insulin response, andgastric emptying.

Experimental Design: The study was divided into two periods and enrolled20 patients diagnosed with type 2 diabetes ranging in age from 20 to 64years of age. Period 1 was an open-label, single-dose, trial in which 15of the patients received a dry powder formulation comprising 1.5 mg ofGLP-1 in FDKP administered after having fasted overnight. As control, 5subjects received FDKP inhalation powder after fasting overnight. Period2 was performed after completion of Period 1. In this part of the study,the patients were given 4 sequential treatments each with a mealchallenge consisting of 475 Kcal and labeled with ¹³C-octanoate asmarker. The study was designed as a double-blind, double dummy,cross-over, meal challenge study, in which saline as control andexenatide were given as injection 15 minutes before a meal and drypowder formulations of inhalable GLP-1 or placebo consisting of a drypowder formulation without GLP-1, were administered immediately beforethe meal and repeated 30 minutes after the meal. The four treatmentswere as follows: Treatment 1 consisted of all patients receiving aplacebo of 1.5 mg of dry powder formulation without GLP-1. In Treatment2, all patients received one dose of 1.5 mg of GLP-1 in a dry powderformulation comprising FDKP. In Treatment 3, all patients received twodoses of 1.5 mg of GLP-1 in a dry powder formulation comprising FDKP,one dose immediately before the meal and one dose 30 minutes after themeal. In Treatment 4, the patients received 10 μg of exenatide bysubcutaneous injection. Blood samples from each patient were taken atvarious times before and after dosing and analyzed for severalparameters, including GLP-1 concentration, insulin response, glucoseconcentration and gastric emptying. The results of this study aredepicted in FIGS. 18-20.

FIG. 18 depicts the mean GLP-1 levels in blood by treatment group asdescribed above. The data demonstrate that the patients receiving thedry powder formulation comprising 1.5 mg of GLP-1 in FDKP hadsignificantly higher levels of GLP-1 in blood soon after administrationas shown in panels A, B and C and that the levels of GLP-1 sharplydeclined after administration in fed or fasted individuals. There wereno measurable levels of GLP-1 in the exenatide-treated group (Panel D),or in controls (Panel E) receiving the dry powder formulation.

FIG. 19 depicts the insulin levels of the patients in the study beforeor after treatment. The data show that endogenous insulin was producedin all patients after treatment including the placebo-treated patientsin the meal challenge studies (Panel B), except for the fasted controlpatients (Panel C) who received the placebo. However, the insulinresponse was more significant in patients receiving GLP-1 in a drypowder composition comprising FDKP, in which the insulin response wasobserved immediately after treatment in both fed and fasted groups(Panels D-F). The results also showed that the glucose levels werereduced in patients treated with the dry powder formulation of GLP-1.Administration of the dry powder formulation of GLP-1 resulted in adelayed rise in blood glucose and reduced overall exposure (AUC) toglucose. Both the delayed rise and lessened exposure were morepronounced in subjects receiving a second administration of GLP-1inhalation powder (data not shown). The magnitude of insulin releasevaried among patients, with some showing small but physiologicallyrelevant levels of insulin whereas others exhibited much larger insulinreleases. Despite the difference in insulin response between thepatients, the glucose response was similar. This difference in insulinresponse may reflect variations in degree of insulin resistance anddisease progression. Assessment of this response can be used as adiagnostic indicator of disease progression with larger releases(lacking greater effectiveness at controlling blood glucose levels)indicating greater insulin resistance and disease progression.

FIG. 20 depicts the percent gastric emptying by treatment groups. PanelA (patients in Treatment 3) and Panel B (patients in Treatment 2)patients had similar gastric emptying characteristics or percentages asthe control patients shown in Panel D (Placebo treated patients with adry powder formulation comprising FDKP without GLP-1). The data alsoshow that patients treated with exenatide even at a 10 μg dose exhibiteda significant delay or inhibition in gastric emptying when compared tocontrols. The data also demonstrate that the present system fordelivering active agents comprising FDKP and GLP-1 lacks inhibition ofgastric emptying; induces a rapid insulin release following GLP-1delivery and causes a reduction in glucose AUC levels.

While the invention has been particularly shown and described withreference to particular embodiments, it will be appreciated thatvariations of the above-disclosed and other features and functions, oralternatives thereof, may be desirably combined into many otherdifferent systems or applications. Also that various presentlyunforeseen or unanticipated alternatives, modifications, variations orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the following claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A method for the treatment of hyperglycemia or diabetes in a patient,comprising the step of administering prandially to a patient in need oftreatment an inhalable rapid-acting dry powder GLP-1 formulation, thedry powder formulation comprising microparticles comprising atherapeutically effective amount of a GLP-1 molecule in an amount of atleast about 0.5% by weight and a diketopiperazine, wherein the weightratio of diketopiperazine to GLP-1 is at least about 17:3, and adipeptidyl peptidase-IV (DPP-IV) inhibitor, and wherein the GLP-1formulation comprises about 0.5 mg to about 3 mg of GLP-1 dose.
 2. Themethod of claim 1, wherein the patient is a mammal suffering with Type 2diabetes mellitus.
 3. The method of claim 1, wherein thediketopiperazine is 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; whereinX is selected from the group consisting of succinyl, glutaryl, maleyl,and fumaryl; or a pharmaceutically acceptable salt thereof.
 4. Themethod of claim 1, wherein the GLP-1 molecule is selected from the groupconsisting of a native GLP-1, a GLP-1 analog, a GLP-1 derivative, a longacting GLP-1 analog, a GLP-1 mimetic, and combinations thereof.
 5. Themethod of claim 1, further comprising administering to a patient atherapeutically amount of an insulin molecule.
 6. The method of claim 5,wherein the inhalable dry powder formulation comprises the GLP-1molecule co-formulated with the insulin molecule.
 7. The method of claim5, wherein the insulin molecule is administered separately as aninhalable dry powder formulation.
 8. The method of claim 1, wherein theinhalable dry powder formulation lacks inhibition of gastric emptying.9. A method for reducing glucose levels in a Type 2 diabetic patientsuffering from hyperglycemia, the method comprising the step ofadministering to said patient in need of treatment an inhalablerapid-acting dry powder GLP-1 formulation for pulmonary administration,said dry powder inhalable formulation comprising microparticlescomprising a therapeutically effective amount of GLP-1 in an amount ofat least about 0.5% by weight and a diketopiperazine or pharmaceuticallyacceptable salt thereof, wherein the weight ratio of diketopiperazine toGLP-1 is at least about 17:3, and a DPP-IV inhibitor, and wherein theGLP-1 formulation comprises about 0.02 mg to about 2 mg of GLP-1 dose.10. The method of claim 9, wherein the glucose levels are reduced byabout 0.1 mmol/L to about 3 mmol/L for a period of approximately fourhours after administration of said inhalable formulation to saidpatient.
 11. The method of claim 9, wherein the inhalable formulation isadministered to said Type 2 diabetic patient prandially, preprandially,prandially, post-prandially or in a fasting state.
 12. The method ofclaim 9, wherein the diketopiperazine is2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl; or apharmaceutically acceptable salt thereof.
 13. The method of claim 9,wherein the inhalable dry powder formulation comprises the GLP-1molecule co-formulated with an insulin molecule.
 14. The method of claim9, wherein the method further comprises administering insulin as aninhalable dry powder formulation.
 15. The method of claim 14, whereinthe insulin is a rapid acting or a long acting insulin.
 16. The methodof claim 9, further comprising administering a formulation comprising along acting GLP-1 analog.
 17. The method of claim 9, wherein theinhalable dry powder formulation lacks inhibition of gastric emptying.18. A method for the treatment of hyperglycemia or diabetes in apatient, comprising the step of administering prandially to a patient inneed thereof an inhalable rapid-acting dry powder GLP-1 formulation, thedry powder formulation comprising microparticles comprising atherapeutically effective amount of a GLP-1 molecule and adiketopiperazine, and a DPP-IV inhibitor; wherein the weight ratio ofdiketopiperazine to GLP-1 is at least about 17:3, wherein said GLP-1reaches a peak plasma concentration greater than 100 pMol/L by 6 minutesafter administration, and wherein the GLP-1 formulation comprises about0.5 mg to about 3 mg of GLP-1 dose.
 19. The method of claim 18, whereinthe GLP-1 and the DPP-IV inhibitor are administered in separatecompositions.
 20. The method of claim 18, wherein the GLP-1 and theDPP-IV inhibitor are administered in a single composition.